Polyamine Derivatives

ABSTRACT

Disclosed are compounds, compositions and methods for systemic and local delivery of biologically active molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Application No.61/161,828, filed Mar. 20, 2009, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to symmetrical polyamine derivatives andformulations comprising such compounds, and more specifically topolyamine derivatives that are partially acylated and optionally carryan auxiliary group. Such compounds are useful in introducing siRNA intoa cell, in silencing expression of a target sequence, in delivering invivo of siRNA, and in treating diseases and/or disorders.

2. Description of the Related Art

The compounds, compositions and methods of the invention are useful intherapeutic, research, and diagnostic applications that rely upon theefficient transfer of biologically active molecules into cells, tissues,and organs. The discussion is provided only for understanding of theinvention that follows.

The cellular delivery of various therapeutic compounds, such asantiviral and chemotherapeutic agents, is usually compromised by twolimitations. First, the selectivity of a number of therapeutic agents isoften low, resulting in high toxicity to normal tissues. Secondly, thetrafficking of many compounds into living cells is highly restricted bythe complex membrane systems of the cell. Specific transporters allowthe selective entry of nutrients or regulatory molecules, whileexcluding most exogenous molecules such as nucleic acids and proteins.Various strategies can be used to improve transport of compounds intocells, including the use of lipid carriers, biodegradable polymers, andvarious conjugate systems.

The most well studied approaches for improving the transport of foreignnucleic acids into cells involve the use of viral vectors or cationiclipids and related cytofectins. Viral vectors can be used to transfergenes efficiently into some cell types, but they generally cannot beused to introduce chemically synthesized molecules into cells. Analternative approach is to use delivery formulations incorporatingcationic lipids, which interact with nucleic acids through one end andlipids or membrane systems through another. Synthetic nucleic acids aswell as plasmids can be delivered using the cytofectins, although theutility of such compounds is often limited by cell-type specificity,requirement for low serum during transfection, and toxicity.

Another approach to delivering biologically active molecules involvesthe use of conjugates. Conjugates are often selected based on theability of certain molecules to be selectively transported into specificcells, for example via receptor-mediated endocytosis. By attaching acompound of interest to molecules that are actively transported acrossthe cellular membranes, the effective transfer of that compound intocells or specific cellular organelles can be realized. Alternatively,molecules that are able to penetrate cellular membranes without activetransport mechanisms, for example, various lipophilic molecules, can beused to deliver compounds of interest. Examples of molecules that can beutilized as conjugates include but are not limited to peptides,hormones, fatty acids, vitamins, flavonoids, sugars, reporter molecules,reporter enzymes, chelators, porphyrins, intercalators, and othermolecules that are capable of penetrating cellular membranes, either byactive transport or passive transport.

The delivery of compounds to specific cell types, for example, cancercells or cells specific to particular tissues and organs, can beaccomplished by utilizing receptors associated with specific cell types.Particular receptors are overexpressed in certain cancerous cells,including the high affinity folic acid receptor. For example, the highaffinity folate receptor is a tumor marker that is overexpressed in avariety of neoplastic tissues, including breast, ovarian, cervical,colorectal, renal, and nasoparyngeal tumors, but is expressed to a verylimited extent in normal tissues. The use of folic acid based conjugatesto transport exogenous compounds across cell membranes can provide atargeted delivery approach to the treatment and diagnosis of disease andcan provide a reduction in the required dose of therapeutic compounds.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates, including folate bioconjugates. The synthesis ofbiologically active pteroyloligo-L-glutamates has been reported. Amethod for the solid phase synthesis of certain oligonucleotide-folateconjugates has been described, as well as oligonucleotides modified withspecific conjugate groups. The use of biotin and folate conjugates toenhance transmembrane transport of exogenous molecules, includingspecific oligonucleotides has been reported. Certain folate conjugates,including specific nucleic acid folate conjugates with a phosphoramiditemoiety attached to the nucleic acid component of the conjugate, andmethods for the synthesis of these folate conjugates have beendescribed. The synthesis of an intermediate,alpha-[2-(trimethylsilyl)ethoxycarbonyl]folic acid, useful in thesynthesis of certain types of folate-nucleoside conjugates has beenreported.

The delivery of compounds to other cell types can be accomplished byutilizing receptors associated with a certain type of cell, such ashepatocytes. For example, drug delivery systems utilizingreceptor-mediated endocytosis have been employed to achieve drugtargeting as well as drug-uptake enhancement. The asialoglycoproteinreceptor (ASGPr) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (example of this high specificity through the useof N-acetyl-D-galactosamine as the carbohydrate moiety, which has higheraffinity for the receptor, compared to galactose). This “clusteringeffect” has also been described for the binding and uptake ofmannosyl-terminating glycoproteins or glycoconjugates. The use ofgalactose and galactosamine based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto the treatment of liver disease such as HBV and HCV infection orhepatocellular carcinoma. The use of bioconjugates can also provide areduction in the required dose of therapeutic compounds required fortreatment. Furthermore, therapeutic bioavailability, pharmacodynamics,and pharmacokinetic parameters can be modulated through the use ofbioconjugates.

A number of peptide based cellular transporters have been developed byseveral research groups. These peptides are capable of crossing cellularmembranes in vitro and in vivo with high efficiency. Examples of suchfusogenic peptides include a 16-amino acid fragment of the homeodomainof ANTENNAPEDIA, a Drosophila transcription factor; a 17-mer fragmentrepresenting the hydrophobic region of the signal sequence of Kaposifibroblast growth factor with or without NLS domain; a 17-mer signalpeptide sequence of caiman crocodylus Ig(5) light chain; a 17-amino acidfusion sequence of HIV envelope glycoprotein gp4114; the HIV-1 Tat49-57fragment; a transportan A-achimeric 27-mer consisting of N-terminalfragment of neuropeptide galanine and membrane interacting wasp venompeptide mastoporan; and a 24-mer derived from influenza virushemagglutinin envelope glycoprotein. These peptides were successfullyused as part of an antisense oligodeoxyribonucleotide-peptide conjugatefor cell culture transfection without lipids. In a number of cases, suchconjugates demonstrated better cell culture efficacy then parentoligonucleotides transfected using lipid delivery. In addition, use ofphage display techniques has identified several organ targeting andtumor targeting peptides in vivo. Conjugation of tumor targetingpeptides to doxorubicin has been shown to significantly improve thetoxicity profile and has demonstrated enhanced efficacy of doxorubicinin the in vivo murine cancer model MDA-MB-435 breast carcinoma.

Another approach to the intracellular delivery of biologically activemolecules involves the use of cationic polymers (for example, the use ofhigh molecular weight lysine polymers for increasing the transport ofvarious molecules across cellular membranes has been described). Certainmethods and compositions for transporting drugs and macromoleculesacross biological membranes in which the drug or macromolecule iscovalently attached to a transport polymer consisting of from 6 to 25subunits, at least 50% of which contain a guanidine or amidine sidechain have been disclosed. The transport polymers are preferablypolyarginine peptides composed of all D-, all L- or mixtures of D- andL-arginine. Described also are certain poly-lysine and poly-argininecompounds for the delivery of drugs and other agents across epithelialtissues, including the skin, gastrointestinal tract, pulmonaryepithelium and blood-brain barrier. Certain polyarginine compounds andcertain poly-lysine and poly-arginine compounds for intra-oculardelivery of drugs have also been disclosed. Certain cyclodextranpolymers compositions that include a cross-linked cationic polymercomponent and certain lipid based formulations have been disclosed.

Another approach to the intracellular delivery of biologically activemolecules involves the use of liposomes or other particle formingcompositions. Since the first description of liposomes in 1965, therehas been a sustained interest and effort in the area of developinglipid-based carrier systems for the delivery of pharmaceutically activecompounds. Liposomes are attractive drug carriers since they protectbiological molecules from degradation while improving their cellularuptake. One of the most commonly used classes of liposome formulationsfor delivering polyanions (e.g., DNA) is that which contains cationiclipids. Lipid aggregates can be formed with macromolecules usingcationic lipids alone or including other lipids and amphiphiles such asphosphatidylethanolamine. It is well known in the art that both thecomposition of the lipid formulation as well as its method ofpreparation have effect on the structure and size of the resultantanionic macromolecule-cationic lipid aggregate. These factors can bemodulated to optimize delivery of polyanions to specific cell types invitro and in vivo. The use of cationic lipids for cellular delivery ofbiologically active molecules has several advantages. The encapsulationof anionic compounds using cationic lipids is essentially quantitativedue to electrostatic interaction. In addition, it is believed that thecationic lipids interact with the negatively charged cell membranesinitiating cellular membrane transport.

Experiments have shown that plasmid DNA can be encapsulated in smallparticles that consist of a single plasmid encapsulated within a bilayerlipid vesicle. These particles typically contain the fusogenic lipiddioleoylphosphatidylethanolamine (DOPE), low levels of a cationic lipid,and can be stabilized in aqueous media by the presence of apoly(ethylene glycol) (PEG) coating. These particles have systemicapplications as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection, can accumulate preferentially in varioustissues and organs or tumors due to the enhanced vascular permeabilityin such regions, and can be designed to escape the lyosomic pathway ofendocytosis by disruption of endosomal membranes. These properties canbe useful in delivering biologically active molecules to various celltypes for experimental and therapeutic applications. For example, theeffective use of nucleic acid technologies such as short interfering RNA(siRNA), antisense, ribozymes, decoys, triplex forming oligonucleotides,2-5A oligonucleotides, and aptamers in vitro and in vivo may benefitfrom efficient delivery of these compounds across cellular membranes.Certain compositions consisting of the combination of siRNA, certainamphipathic compounds, and certain polycations have been disclosed.Certain lipid based formulations, certain lipid encapsulated interferingRNA formulations, and certain polycationic compositions for the cellulardelivery of polynucleotides have been described. Short interferingnucleic acid molecules (siNA) and various technologies for the deliveryof siNA molecules and other polynucleotides have also been described.

In addition, recent work involving cationic lipid particles demonstratedthe formation of two structurally different complexes comprising nucleicacid (or other polyanionic compound) and cationic lipid. One structurecomprises a multilamellar structure with nucleic acid monolayerssandwiched between cationic lipid bilayers (“lamellar structure”). Asecond structure comprises a two dimensional hexagonal columnar phasestructure (“inverted hexagonal structure”) in which nucleic acidmolecules are encircled by cationic lipid in the formation of ahexagonal structure. Authors also demonstrated that the invertedhexagonal structure transfects mammalian cells more efficiently than thelamellar structure. Further, optical microscopy studies showed that thecomplexes comprising the lamellar structure bind stably to anionicvesicles without fusing to the vesicles, whereas the complexescomprising the inverted hexagonal structure are unstable and rapidlyfuse to the anionic vesicles, releasing the nucleic acid upon fusion.

The structural transformation from lamellar phase to inverted hexagonalphase complexes is achieved either by incorporating a suitable helperlipid that assists in the adoption of an inverted hexagonal structure orby using a co-surfactant, such as hexanol. However, neither of thesetransformation conditions are suitable for delivery in biologicalsystems. Furthermore, while the inverted hexagonal complex exhibitsgreater transfection efficiency, it has very poor serum stabilitycompared to the lamellar complex. Thus, there remains a need to designdelivery agents that are serum stable.

SUMMARY OF THE INVENTION

The invention provides compounds, compositions and methods for improvingthe efficiency of systemic and local delivery of biologically activemolecules. Among other things, the invention provides compounds,compositions and methods for making and using delivery agents that arestable in circulation and undergo structural changes under appropriatephysiological conditions (e.g., pH) which increase the efficiency ofdelivery of biologically active molecules.

In a broad aspect, the invention encompasses the compounds of formulaeI-VI, shown below.

Thus, one aspect of the invention provides compounds of formula I

wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen; and-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds.

The second aspect of the invention provides compounds of formula II

wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen; and-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   G₁ and G₂ are independently hydrogen or a polymer moiety.

The third aspect of the invention provides compounds of formula III,

wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   T₁ and T₂ are independently hydrogen or a targeting ligand;-   G₁ and G₂ are independently bond or a polymer moiety,    where at least one of T₁ and T₂ is a targeting ligand.

The fourth aspect of the invention provides compounds of formula IV,

wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   G is hydrogen or a polymer moiety.

The fifth aspect of the invention provides compounds of formula V,

wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   T is a targeting ligand; and-   G is a bond or a polymer moiety.

In another aspect, the invention provides compounds of formula VI,

wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   R_(N) represents NHR₄, NR₄R₅, or N⁺R₄R₅R₆; where    -   R₄, R₅, and R₆ independently represent C₁-C₆ alkyl groups.

The invention also provides synthetic intermediates that are useful inmaking the compounds of Formulae I-VI.

One aspect of the invention provides formulations comprising a compoundof any formulae I-VI. The formulations of the invention containlipoplexes or liposomes formed by the compounds of formulae I-VI.

One aspect of the invention provides formulations comprising a compoundof any formulae I-VI and another molecule, which may be a biologicallyactive molecule. The biologically active molecule may be (a) selectedfrom the group consisting of: ribosomal RNA; antisense polynucleotidesof RNA or DNA; ribozymes; siRNA; shRNA; miRNA; and polynucleotides ofgenomic DNA, cDNA, or mRNA that encode for a therapeutically usefulprotein; or (b) proteins, peptides, cholesterol, hormones, smallmolecules such as antivirals or chemotherapeutics, vitamins, andco-factors.

In a related aspect, the invention provides formulations comprising acompound of any formulae I-VI and an aptamer. In these formulations, theaptamer is not covalently bound to the compound. Thus, where thecompound contains a targeting ligand, T, the resulting formulation mayinclude a covalently bound targeting ligand and a aptamer that is notcovalently bound. In these formulations, the aptamers and targetingligands may be the same or different.

Another aspect of the invention provides formulations comprisingparticles formed by the compounds of any formula I-VI and anothermolecule, which may be a biologically active molecule. In this aspect,the invention provides stable particles useful for encapsulating, e.g.,one or more siRNA molecules.

One aspect of the invention provides a method of introducing siRNA intoa cell, comprising contacting the cell with a formulation of theinvention.

One aspect of the invention provides a method of modulating expressionof a target sequence, said method comprising administering to amammalian subject a therapeutically effective amount of a formulation ofthe invention.

Another aspect of the invention provides a method for in vivo deliveryof siRNA, said method comprising administering to a mammalian subject atherapeutically effective amount of a formulation of the invention.

Another aspect of the invention provides a method for in vivo deliveryof plasmid DNA, said method comprising administering to a mammaliansubject a therapeutically effective amount of a formulation of theinvention.

In yet another aspect of the invention provides a method of treating orpreventing a disease in a mammalian subject, said method comprisingadministering to said subject a therapeutically effective amount of aformulation of the invention.

It has been surprisingly discovered that formulations and deliverysystems made using compounds of the invention, for example, dioleoylmonoamine, are both efficacious for transcript specific knockdown andare relatively non-toxic.

It has also been surprisingly discovered that the use of formulationsand delivery systems of the invention results in preferential uptake ofa drug by lung tissue and also leads to preferential transcriptknockdown in lung relative to other tissues such as liver.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing protein expression levels of VEGF in cellculture medium following an in vitro transfection using murine squamouscell carcinoma VII (SCCVII) cells. Cells were transfected with siRNAformulated with dioleoyl monoamine.

FIG. 2 is a graph showing protein expression levels of VEGF in cellculture medium following an in vitro transfection using murine squamouscell carcinoma VII (SCCVII) cells. Cells were transfected with siRNAformulated with dioleoyl monoamine or methyl-dioleoyl monoamine.

FIG. 3 is a graph showing siRNA specific transcript knockdown(Caveolin-1) in the lung and livers of mice following a single ivinjection of siRNA formulated with dioleoyl monoamine.

FIG. 4A and FIG. 4B are graphs showing mVEGF transcript levels in SCCVIItumors following IT injection of dioleoyl monoamine formulated VEGFsiRNA (FIG. 4A) and inhibition of tumor growth (FIG. 4B) in micefollowing intratumoral administration of formulated VEGF siRNA.

FIG. 5A and FIG. 5B are graphs showing relative transcript levels ofCaveolin-1 (Cav-1) in cell culture medium following an in vitrotransfection using murine squamous cell carcinoma VII (SCCVII) cells.Cells were transfected with siRNA and dioleoyl monoamine/mPEG-dioleoylmonoamine complexes (FIG. 5A) or encapsulated siRNA with dioleoylmonoamine/mPEG-dioleoyl monoamine (FIG. 5B).

FIG. 6 shows dose dependent (10 μg-100 μg), siRNA specific transcriptknockdown (Cav-1) in the lung of mice following a single iv injection ofsiRNA complexed with dioleoyl monoamine and mPEG-dioleoyl monoamine.

FIG. 7 shows protein expression levels of β-actin in cell culture mediumfollowing an in vitro transfection using HepG2 cells. Cells weretransfected with siRNA formulated with dioleoylmonoamine/lactobionyl-dioleoyl monoamine.

FIG. 8 shows controlled-release of Cav-1 siRNA complexed with dioleoylmonoamine from alginate gels with and without EDTA.

FIG. 9 shows siRNA specific transcript knockdown (Cav-1) in the lung andliver of mice following a single iv injection of siRNA complexed withdioleoyl monoamine and mPEG-dioleoyl monoamine, or with DOTAP:DOPE(1:1), or with BPEI.

FIG. 10 shows transfection activity of encapsulated siRNA with dioleoylcrossamine/prostate specific membrane antigen (PSMA) targeting aptamer.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention provides compounds of formula I,

and the pharmaceutically acceptable salts thereof, wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen; and-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds.

In one embodiment, the invention provides compounds of formula I whereinn1, n2, n3, and n4 are all 1, and X and X′ are bonds.

In one embodiment, the invention provides compounds of formula I whereinat least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon group containing from1-4 double bonds.

In another embodiment, the invention provides compounds of formula Iwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In yet another embodiment, the invention provides compounds of formula Iwherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing1 or 2 double bonds.

In one embodiment, the invention provides compounds of formula I whereinR₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing 1double bond.

In one embodiment, the invention provides compounds of formula I whereinR₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groups containing 1 or 2double bonds.

In yet another embodiment, the invention provides compounds of formulaI, wherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula I whereinboth of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In another embodiment, the invention provides compounds of formula Iwherein n1, n2, n3, and n4 are the same and are 1 or 2; X and X′ arebonds; and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In still another embodiment, the invention provides compounds of formulaI wherein n1, n2, n3, and n4 are the same and are 1 or 2; X and X′ arebonds; and R₁ and R₂ are the same and represent C₈-C₂₅ hydrocarbongroups containing from 1-4 double bonds.

In yet another embodiment, the invention provides compounds of formula Iwherein n1, n2, n3, and n4 are the same and are 1 or 2; X and X′ arebonds; and R₁ and R₂ are the same and represent a C₈-C₂₅ hydrocarbongroup containing from 1-2, preferably 1, double bonds.

In yet another embodiment, the invention provides compounds of formula Iwherein n1, n2, n3, and n4 are 1; X and X′ are bonds; and R₁ and R₂ arethe same and represent a C₁₄-C₂₀ hydrocarbon group containing from 1-2,preferably 1, double bonds.

Another aspect of the invention provides compounds of formula II,

wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen; and-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   G₁ and G₂ are independently hydrogen or a polymer moiety.

In one embodiment, the invention provides compounds of formula IIwherein n1, n2, n3, and n4 are all 1, and X and X′ are both bonds.

In one embodiment, the invention provides compounds of formula IIwherein at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaII wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formulaII wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula IIwherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formulaII wherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula IIwherein both of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In one embodiment, the invention provides compounds of formula II whereone of G₁ and G₂ is a polymer moiety and the other is hydrogen.

In one embodiment, the invention provides compounds of formula IIwherein one of G₁ and G₂ is a polyoxyalkylene, polyvinylpyrrolidone,polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl)methacrylamide, or polydivinylether maleic anhydride.

In another embodiment, the invention provides compounds of formula IIwherein the polymer comprises at least one linker group between polymerunits.

In yet another embodiment, the invention provides compounds of formulaII where the polymer moiety is a polyoxyalkylene.

In yet another embodiment, the invention provides compounds of formulaII where the molecular weight of the polymer is from about 200-10,000Da. Preferred polymers have molecular weights ranging from about1,000-5,000 Da.

In one embodiment, the invention provides compounds of formula II wherethe polymer moiety comprises at least one linker selected from —C(O)—,—O—, —O—C(O)O—, —C(O)CH₂CH₂C(O)—. —S—S—, —NR³—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—, -alkylene-NR³C(O)O—,-alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—, -alkylene-NR³—,-alkylene-O—, -alkylene-NR³C(O)—, -alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O -alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,-alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR³-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, and -alkyleneoxy-NR³C(O)O-alkyleneoxy-, where R³is hydrogen, or optionally substituted alkyl, and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—,-alkylene-OC(O)NR³-alkylene-NR³-alkylene-O—, -alkylene-NR³C(O)—,alkylene-C(O)NR³—, —NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-,—OC(O)NR³-alkylene-, —NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O-alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

In one embodiment, the invention provides compounds of formula IIwherein the polymer is a polyoxyethylene where the oxyalkylene groupsare independently straight or branched chain polyoxyalkylene groupshaving from 2-5 carbon atoms in their repeating units.

In one embodiment, the invention provides compounds of formula IIwherein the polyoxyalkylene is a polyoxyethylene, a straight or branchedchain polyoxypropylene, or a straight or branched chain polyoxybutylene.

In one embodiment, the invention provides compounds of formula IIwherein the polyoxyalkylene is a polyoxyethylene.

In another embodiment, the invention provides compounds of formula IIwhere n1, n2, n3, and n4 are all the same and are 1 or 2; X and X′ areboth bonds; and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIwhere n1, n2, n3, and n4 are all the same and are 1 or 2; X and X′ areboth bonds; and R₁ and R₂ are identical and represent C₈-C₂₅ hydrocarbongroups containing from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIwhere n1, n2, n3, and n4 are all the same and are 1; X and X′ are bothbonds; and R₁ and R₂ are identical and represent C₈-C₂₅ hydrocarbongroups containing from 1-4 double bonds.

In still another embodiment, the invention provides compounds of formulaII where n1, n2, n3, and n4 are all the same and are 1; X and X′ areboth bonds; and R₁ and R₂ are identical and represent C₁₄-C₂₀hydrocarbon groups containing from 1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaII where n1, n2, n3, and n4 are all the same and are 1; X and X′ areboth bonds; and R₁ and R₂ are identical and represent C₁₄-C₂₀hydrocarbon groups containing 1 or 2 double bonds.

Another aspect of the invention provides compounds of formula III,

wherein

-   n1, n2, n3, and n4 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   T₁ and T₂ are independently hydrogen or a targeting ligand;-   G₁ and G₂ are independently bond or a polymer moiety, where at least    one of T₁ and T₂ is a targeting ligand.

In one embodiment, the invention provides compounds of formula IIIwherein n1, n2, n3, and n4 are all 1, and both X and X′ are bonds.

In one embodiment, the invention provides compounds of formula IIIwherein at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In one embodiment, the invention provides compounds of formula IIIwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIIwherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formulaIII wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 double bond.

In yet another embodiment, the invention provides compounds of formulaIII wherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 or 2 double bonds.

In one embodiment, the invention provides compounds of formula IIIwherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula IIIwherein both of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In one embodiment, the invention provides compounds of formula III whereone of G₁ and G₂ is a polymer moiety and the other is hydrogen.

In yet another embodiment, the invention provides compounds of formulaIII wherein the one of G₁ and G₂ is a polyoxyalkylene,polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinylalcohol, dextran, poly (L-glutamic acid), styrene maleic anhydride,poly-N-(2-hydroxypropyl) methacrylamide, or polydivinylether maleicanhydride.

In another embodiment, the invention provides compounds of formula IIIwherein the polymer comprises at least one linker group between polymerunits.

In yet another embodiment, the invention provides compounds of formulaIII where the polymer moiety is a polyoxyalkylene.

In one embodiment, the invention provides compounds of formula III wherethe molecular weight of the polymer is from about 200-10,000 Da.Preferred polymers have molecular weights ranging from about 1,000-5,000Da.

In one embodiment, the invention provides compounds of formula III wherethe polymer moiety comprises at least one linker selected from —C(O)—,—O—, —O—C(O)O—, —C(O)CH₂CH₂C(O)—, —S—S—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—, -alkylene-NR³C(O)O—,-alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—, -alkylene-NR³—,-alkylene-O—, -alkylene-NR³C(O)—, -alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,-alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-,—OC(O)NR³-alkyleneoxy-, —NR³-alkyleneoxy-, —O-alkyleneoxy-,—NR³C(O)-alkyleneoxy-, —C(O)NR³-alkyleneoxy-, and-alkyleneoxy-NR³C(O)O-alkyleneoxy-, where R³ is as defined above and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,-alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—, alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene-,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O -alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

In one embodiment, the invention provides compounds of formula IIIwherein the polymer is a polyoxyethylene where the oxyalkylene groupsare independently straight or branched chain polyoxyalkylene groupshaving from 2-5 carbon atoms in their repeating units.

In another embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene, a straight or branchedchain polyoxypropylene, or a straight or branched chain polyoxybutylene.

In one embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is a pharmacologically active small molecule, an endosomolyticagent, a fusogenic peptide, a cell membrane permeating agent, a chargemasking agent, a nucleic acid, or a cell receptor ligand.

In one embodiment, the invention provides compounds of formula IIIwherein the targeting ligand is a pharmacologically active smallmolecule that has anti-proliferative activity.

In one embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is a pharmacologically active small molecule that hasanti-proliferative activity.

In one embodiment, the invention provides compounds of formula IIIwherein the targeting ligand is a folic acid group.

In one embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is a folic acid group.

In one embodiment, the invention provides compounds of formula IIIwherein the targeting ligand is a fusogenic peptide.

In one embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is a fusogenic peptide.

In one embodiment, the invention provides compounds of formula IIIwherein the targeting ligand is selected from the group consisting ofbiotin, galactose, acetylsalicylic acid, naproxen, and a cell receptorligand.

In one embodiment, the invention provides compounds of formula IIIwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is selected from the group consisting of biotin, galactose,acetylsalicylic acid, naproxen, and a cell receptor ligand.

In another embodiment, the invention provides compounds of formula IIIwhere n1, n2, n3, and n4 are the same and are 1 or 2; both X and X′ arebonds; and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIIwhere n1, n2, n3, and n4 are the same and are 1 or 2; both X and X′ arebonds; and R₁ and R₂ are the same and represent a C₈-C₂₅ hydrocarbongroup containing from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIIwhere n1, n2, n3, and n4 are 1; both X and X′ are bonds; and R₁ and R₂are the same and represent a C₈-C₂₅ hydrocarbon group containing from1-4 double bonds.

In another embodiment, the invention provides compounds of formula IIIwhere n1, n2, n3, and n4 are 1; both X and X′ are bonds; and R₁ and R₂are the same and represent a C₈-C₂₅ hydrocarbon group containing from1-2 double bonds.

In another embodiment, the invention provides compounds of formula IIIwhere n1, n2, n3, and n4 are 1; both X and X′ are bonds; and R₁ and R₂are the same and represent a C₁₄-C₂₀ hydrocarbon group containing 1double bond.

One aspect of the invention provides compounds of formula IV,

and the pharmaceutically acceptable salts thereof, wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   G is hydrogen or a polymer moiety.

In one embodiment, the invention provides compounds of formula IVwherein n1, n2, and n3 are all 1, and X and X′ are bonds.

In one embodiment, the invention provides compounds of formula IVwherein at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IVwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaIV wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 or 2 double bonds.

In another embodiment, the invention provides compounds of formula IVwherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing1 double bond.

In one embodiment, the invention provides compounds of formula IVwherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 or 2 double bonds.

In another embodiment, the invention provides compounds of formula IVwherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula IVwherein both of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In one embodiment, the invention provides compounds of formula IV whereX is oxygen.

In one embodiment, the invention provides compounds of formula IV whereX is nitrogen.

In one embodiment, the invention provides compounds of formula IVwherein G is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide,polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamicacid), styrene maleic anhydride, poly-N-(2-hydroxypropyl)methacrylamide, or polydivinylether maleic anhydride.

In another embodiment, the invention provides compounds of formula IVwherein the polymer comprises at least one linker group between polymerunits.

In yet another embodiment, the invention provides compounds of formulaIV where the polymer moiety is a polyoxyalkylene.

In yet another embodiment, the invention provides compounds of formulaIV where the molecular weight of the polymer is from about 200-10,000Da. Preferred polymers have molecular weights ranging from about1,000-5,000 Da.

In one embodiment, the invention provides compounds of formula IV wherethe polymer moiety comprises at least one linker selected from —C(O)—,—O—, —O—C(O)O—, —C(O)CH₂CH₂C(O)—, —S—S—, —NR³—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—, -alkylene-NR³C(O)O—,-alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—, -alkylene-NR³—,-alkylene-O—, -alkylene-NR³C(O)-alkylene-, —C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,-alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR³-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, and -alkyleneoxy-NR³C(O)O-alkyleneoxy-, where R³is as defined above and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,-alkylene-NR³-alkylene-O—, -alkylene-NR³C(O)—, alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene-,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O-alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

In one embodiment, the invention provides compounds of formula IVwherein the polymer is a polyoxyethylene where the oxyalkylene groupsare independently straight or branched chain polyoxyalkylene groupshaving from 2-5 carbon atoms in their repeating units.

In another embodiment, the invention provides compounds of formula IVwherein the polyoxyalkylene is a polyoxyethylene, a straight or branchedchain polyoxypropylene, or a straight or branched chain polyoxybutylene.

In yet another embodiment, the invention provides compounds of formulaIV wherein the polyoxyalkylene is a polyoxyethylene.

In another embodiment, the invention provides compounds of formula IVwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon group containingfrom 1-4 double bonds.

In another embodiment, the invention provides compounds of formula IVwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from 1-4double bonds.

In another embodiment, the invention provides compounds of formula IVwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and R₁ and R₂ are the same and represent C₈-C₂₅ hydrocarbon groupscontaining from 1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaIV wherein n1, n2, and n3 are the same and are 1 or 2; X and X′ arebonds; and R₁ and R₂ are the same and represent C₈-C₂₅ hydrocarbongroups containing from 1-2 double bonds.

In yet another embodiment, the invention provides compounds of formulaIV wherein n1, n2, and n3 are 1; X and X′ are bonds; and R₁ and R₂ arethe same and represent C₁₄-C₂₀ hydrocarbon groups containing 1 doublebond.

Another aspect of the invention provides compounds of formula V,

wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   T is a targeting ligand; and-   G is a bond, or a polymer moiety.

In one embodiment, the invention provides compounds of formula V whereinn1, n2, and n3 are all 1, and both X and X′ are bonds.

In one embodiment, the invention provides compounds of formula V whereinat least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon group containing from1-4 double bonds.

In another embodiment, the invention provides compounds of formula Vwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In yet another embodiment, the invention provides compounds of formula Vwherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formula Vwherein R₅ and R₂ are independently C₈-C₂₅ hydrocarbon groups containing1 double bond.

In one embodiment, the invention provides compounds of formula V whereinR₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groups containing 1 or 2double bonds.

In yet another embodiment, the invention provides compounds of formula Vwherein R₅ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula V whereinboth of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In another embodiment, the invention provides compounds of formula Vwherein G is a polymer moiety.

In yet another embodiment, the invention provides compounds of formula Vwherein G is a polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide,polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamicacid, styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide,or polydivinylether maleic anhydride.

In yet another embodiment, the invention provides compounds of formula Vwherein the polymer comprises at least one linker group between polymerunits.

In one embodiment, the invention provides compounds of formula V wherethe polymer moiety is a polyoxyalkylene.

In another embodiment, the invention provides compounds of formula Vwhere the molecular weight of the polymer is from about 200-10,000 Da.The preferred molecular weight of the polymer is from about 1,000-5,000Da.

In one embodiment, the invention provides compounds of formula V wherethe polymer moiety comprises at least one linker selected from —C(O)—,—O—, —O—C(O)O—, —C(O)CH₂CH₂C(O)—, —S—S—, —NR³—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—, -alkylene-NR³C(O)O—,-alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—, -alkylene-NR³—,-alkylene-O—, -alkylene-NR³C(O)—, -alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O -alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,-alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR³-alkylene-,—NR³C(O)O -alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-,—OC(O)NR³-alkyleneoxy, —NR³-alkyleneoxy-, —O-alkyleneoxy-,—NR³C(O)-alkyleneoxy-, —C(O)NR³-alkyleneoxy-, and-alkyleneoxy-NR³C(O)O-alkyleneoxy-, where R³ is as defined above and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—, alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene-,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O -alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-—OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

In another embodiment, the invention provides compounds of formula Vwherein the polymer is a polyoxyethylene where the oxyalkylene groupsare independently straight or branched chain polyoxyalkylene groupshaving from 2-5 carbon atoms in their repeating units.

In yet another embodiment, the invention provides compounds of formula Vwherein the polyoxyalkylene is a polyoxyethylene, a straight or branchedchain polyoxypropylene, or a straight or branched chain polyoxybutylene.

In one embodiment, the invention provides compounds of formula V whereinthe polyoxyalkylene is a polyoxyethylene and the targeting ligand is apharmacologically active small molecule, an endosomolytic agent, afusogenic peptide, a cell membrane permeating agent, a charge maskingagent, or a nucleic acid.

In one embodiment, the invention provides compounds of formula V whereinthe targeting ligand is a pharmacologically active small molecule thathas anti-proliferative activity.

In one embodiment, the invention provides compounds of formula V whereinthe polyoxyalkylene is a polyoxyethylene and the targeting ligand is apharmacologically active small molecule that has anti-proliferativeactivity.

In yet another embodiment, the invention provides compounds of formula Vwherein the targeting ligand is a folic acid group.

In yet another embodiment, the invention provides compounds of formula Vwherein the polyoxyalkylene is a polyoxyethylene and the targetingligand is a folic acid group.

In one embodiment, the invention provides compounds of formula V whereinthe targeting ligand is a fusogenic peptide.

In one embodiment, the invention provides compounds of formula V whereinthe polyoxyalkylene is a polyoxyethylene and the targeting ligand is afusogenic peptide.

In one embodiment, the invention provides compounds of formula V whereinthe targeting ligand is selected from the group consisting of biotin,galactose, acetylsalicylic acid, and naproxen.

In one embodiment, the invention provides compounds of formula V whereinthe polyoxyalkylene is a polyoxyethylene and the targeting ligand isselected from the group consisting of biotin, galactose, acetylsalicylicacid, and naproxen.

In another embodiment, the invention provides compounds of formula Vwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon group containingfrom 1-4 double bonds.

In another embodiment, the invention provides compounds of formula Vwherein n1, n2, n3, and n4 are the same and are 1 or 2; X and X′ arebonds; and both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containingfrom 1-4 double bonds.

In another embodiment, the invention provides compounds of formula Vwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and R₁ and R₂ are the same and represent C₈-C₂₅ hydrocarbon groupscontaining from 1-4 double bonds.

In yet another embodiment, the invention provides compounds of formula Vwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and R₁ and R₂ are the same and represent C₁₄-C₂₀ hydrocarbon groupscontaining from 1-2 double bonds.

In yet another embodiment, the invention provides compounds of formula Vwherein n1, n2, and n3 are 1; X and X′ are bonds; and R₁ and R₂ are thesame and represent C₈-C₂₅ hydrocarbon groups containing 1 double bond.

Another aspect of the invention provides compounds of formula VI

wherein

-   n1, n2, and n3 are independently 1, 2, 3, or 4;-   X and X′ are independently a bond, oxygen, or nitrogen;-   R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groups optionally    containing from 1-4 double or triple bonds; and-   R_(N) represents NHR₄, NR₄R₅, or N¹R₄R₅R₆; where    -   R₄, R₅, and R₆ independently represent C₁-C₆ alkyl groups.

In one embodiment, the invention provides compounds of formula VIwherein n1, n2, and n3 are all 1, and both X and X′ are bonds.

In one embodiment, the invention provides compounds of formula VIwherein at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon groupcontaining from 1-4 double bonds.

In another embodiment, the invention provides compounds of formula VIwherein both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containing from1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaVI wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formulaVI wherein R₁ and R₂ are independently C₈-C₂₅ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula VIwherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 or 2 double bonds.

In yet another embodiment, the invention provides compounds of formulaVI wherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.

In one embodiment, the invention provides compounds of formula VIwherein both of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups.

In another embodiment, the invention provides compounds of formula VIwherein R_(N) represents NHR₄ where R₄ represents a C₁-C₂ alkyl group.

In another embodiment, the invention provides compounds of formula VIwherein R_(N) represents NR₄R₅ where R₄ and R₅ independently representC₁-C₂ alkyl groups.

In another embodiment, the invention provides compounds of formula VIwherein R_(N) represents N⁺R₄R₅R₆ where R₄, R₅, and R₆ independentlyrepresents C₁-C₂ alkyl groups.

In another embodiment, the invention provides compounds of formula VIwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and at least one of R₁ and R₂ is a C₈-C₂₅ hydrocarbon group containingfrom 1-4 double bonds.

In another embodiment, the invention provides compounds of formula VIwherein n1, n2, n3, and n4 are the same and are or 2; X and X′ arebonds; and both of R₁ and R₂ are C₈-C₂₅ hydrocarbon groups containingfrom 1-4 double bonds.

In another embodiment, the invention provides compounds of formula VIwherein n1, n2, and n3 are the same and are 1 or 2; X and X′ are bonds;and R₅ and R₂ are the same and represent C₈-C_(2s) hydrocarbon groupscontaining from 1-4 double bonds.

In yet another embodiment, the invention provides compounds of formulaVI wherein n1, n2, and n3 are the same and are 1 or 2; X and X′ arebonds; and R₁ and R₂ are the same and represent C₁₄-C₂₀ hydrocarbongroups containing from 1-2 double bonds.

In yet another embodiment, the invention provides compounds of formulaVI wherein n1, n2, and n3 are 1; X and X′ are bonds; and R₅ and R₂ arethe same and represent C₈-C₂₅ hydrocarbon groups containing 1 doublebond.

In yet another embodiment, the invention provides compounds of formulaVI wherein n1, n2, and n3 are 1; X and X′ are bonds; R₅ and R₂ are thesame and represent C₈-C₂₅ hydrocarbon groups containing 1 double bond;and R_(N) is represents N⁺R₄R₅R₆ where R₄, R₅, and R₆ independentlyrepresents C₁-C₆ alkyl groups.

In yet another embodiment, the invention provides compounds of formulaVI wherein n1, n2, and n3 are 1; X and X′ are bonds; R₁ and R₂ are thesame and represent C₈-C₂₅ hydrocarbon groups containing 1 double bond;and R_(N) is represents N⁺R₄R₅R₆ where R₄, R₅, and R₆ independentlyrepresents C₁-C₂ alkyl groups.

In another embodiment of compounds of formulae I-VI where X and X′ bothrepresent a bond, suitable C(O)XR₁ and C(O)X′R₂ moieties include groupsderived from saturated, monounsaturated, and polyunsaturated fatty acidshaving from 10-22 carbon atoms, preferably from 12-20 carbon atoms, andmore preferably from 14-18 carbon atoms. Representative C(O)XR₁ andC(O)X′R₂ moieties include those derived from, for example, myristoleicacid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid,arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoicacid, isostearic acid, elaidic acid, petroselinic acid, eleostearicacid, or lauroleic acid.

In another embodiment of compounds of formulae I-VI where X and X′independently both represent a nitrogen or oxygen, suitable R₁ and R₂moieties include groups derived from saturated and polyunsaturated fattyacids having from 10-22 carbon atoms, preferably from 12-20 carbonatoms, and more preferably from 14-18 carbon atoms. Representative R₁and R₂ moieties include those derived from, for example, such as thosederived from myristoleic acid, palmitoleic acid, oleic acid, linoleicacid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucicacid, docosahexaenoic acid, isostearic acid, elaidic acid, petroselinicacid, eleostearic acid, or lauroleic acid.

One aspect of the invention provides formulations comprising a compoundof any of formulae I-VI.

One aspect of the invention provides formulations comprising a compoundof any of formulae I-VI and another molecule, which may be abiologically active molecule, where the molecule is selected from thegroup consisting of: ribosomal RNA; antisense polynucleotides of RNA orDNA; ribozymes; siRNA; shRNA; miRNA; and polynucleotides of genomic DNA,cDNA, or mRNA that encode for a therapeutically useful protein.

Another aspect of the invention provides formulations comprisingparticles formed by the compounds of any of formula I-VI and thebiologically active molecule.

Another aspect of the invention provides a formulation comprising thecompounds of any of formula I-VI and a biologically active molecule,where the biologically active molecule and the compound form a lipoplex.

Another aspect of the invention provides a formulation comprising thecompounds of any of formula I-VI and a biologically active molecule,where the biologically active molecule is at least partially within aliposome formed by the compound.

Another aspect of the invention provides a formulation comprising thecompounds of any of formula I-VI and a biologically active molecule,where the biologically active molecule is encapsulated within theliposome.

In one embodiment, the invention provides formulations where theparticles have a median diameter of less than about 500 nm.

One aspect of the invention provides methods of introducing siRNA into acell, comprising contacting the cell with a formulation of theinvention, where the formulation comprises a compound of any of FormulaeI-VI and a siRNA.

Another aspect of the invention provides methods of introducingsynthetic shRNA into a cell, comprising contacting the cell with aformulation of the invention, where the formulation comprises a compoundof any of Formulae I-VI and a shRNA.

In yet another aspect of the invention provides methods of introducingmiRNA into a cell, comprising contacting the cell with a formulation ofthe invention, where the formulation comprises a compound of any ofFormulae I-VI and a miRNA.

Another aspect of the invention provides methods of introducingantisense nucleic acid into a cell, comprising contacting the cell witha formulation of the invention, where the formulation comprises acompound of any of Formulae I-VI and an antisense nucleic acid.

One aspect of the invention provides methods of modulating expression ofa target sequence, said method comprising administering to a mammaliansubject a therapeutically effective amount of a formulation comprising acompound of any of formulae I-VI and a biologically active molecule.

Another aspect of the invention provides methods for in vivo delivery ofsiRNA, said method comprising administering to a mammalian subject atherapeutically effective amount of a formulation of the invention.

Another aspect of the invention provides methods of treating orpreventing a disease in a mammalian subject, said method comprisingadministering to said subject a therapeutically effective amount of aformulation comprising a compound of any of formulae I-VI and abiologically active molecule.

Another aspect of the invention provides methods for inhibitingexpression of a gene, comprising contacting a cell with a formulationcomprising a biologically active molecule capable of inhibitingexpression of the gene and a compound of any of formulae I-VI. In oneembodiment, the cell is a mammalian cell, preferably a human cell.Further, these methods may be carried out in vivo or in vitro. In oneembodiment, the biologically active molecule is selected from the groupconsisting of, for example, siRNA, shRNA, miRNA, antisense nucleic acid,ribosomal RNA, antisense polynucleotides of RNA or DNA, ribozymes, andpolynucleotides of genomic DNA, cDNA, or mRNA that encode for atherapeutically useful protein. Such formulations may also includecholesterol, hormones, antivirals, peptides, chemotherapeutics, smallmolecules, vitamins, co-factors, or proteins such as antibodies.

In another aspect of the invention, biologically active molecules thatdo not inhibit gene expression, e.g., various small moleculepharmaceutical compounds, can be contained in formulations withcompounds of the invention for use in therapies other than thoseinvolving inhibition of gene expression.

In another embodiment, the invention provides implantable or injectabledevices containing a formulation of (i) one or more of the compounds ofany of formula I-VI and (ii) one or more biologically active molecules.In these devices, the formulation is entrapped or encapsulated withinthe device and the device, in addition to the formulation, comprises abiodegradable and/or biocompatible drug release material. The devicewill be of a size and shape suitable for injection into or implantationwithin the body of a subject, preferably a mammalian, more preferably ahuman, subject.

As noted above, particular compounds of the invention include a polymermoiety, G, G₁, or G₂. These polymer groups have molecular weightsranging from about 200 Da to about 10,000 Da.

The polymer moieties are preferably polyoxyalkylenes or comprise two ormore polyoxyalkylene groups or units. A polyoxyalkylene group is formedby polymerizing alkylene oxide monomers to provide polymer moieties ofdesired size and weight. Where the polymer moiety comprises two or morepolyoxyalkylene groups, the individual polyoxyalkylene groups areconnected to each other by linker groups. Examples of suitable linkergroups are:

-   -   —C(O)—, —O—, —O—C(O)O—, —C(O)CH₂CH₂C(O)—, —S—S—, —NR³—,        —NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,        -alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,        -alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—,        -alkylene-C(O)NR³—, —NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-,        —OC(O)NR³-alkylene, —NR³-alkylene-, —O-alkylene-,        —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,        -alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-,        -alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,        -alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-,        —C(O)NR³-alkylene-, —NR³C(O)O-alkyleneoxy-,        —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,        —NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,        —C(O)NR³-alkyleneoxy-, and -alkyleneoxy-NR³C(O)O-alkyleneoxy-,        where R³ is hydrogen, or optionally substituted alkyl, and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,-alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—, -alkylene-C(O)NR³—,—NR³C(O)O -alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene-,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O-alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, -alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

Preferred linker groups are —C(O)—, —O—, —NR³—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—, and —C(O)NR³—, where each R³ is as defined above.

Where G, G₁, or G₂ is formed from independent units linked by, forexample, amide groups, the units may be selected from shorter chainpolymers or units having a wide range of sizes and molecular weights. Asnoted above, polymers G, G₁, and G₂, may have molecular weights of fromabout 200-10,000 Da; any of these polymers may be formed from severalshorter, independently-sized units. The units may have molecular weightsindependently ranging from about 50 (i.e., one repeating unit of apolyethylene glycol), 200, or 500 Da up to about 3000, 4000 or 5000 Da.

Thus, where a compound includes a polymer moiety G, G₁, or G₂, having amolecular weight of about 3000 Da, the polymer may, for example,

-   -   (a) be a polyoxyethylene group having a molecular weight of        about 3000 Da;    -   (b) consist of four polyoxyethylene groups covalently bound to        each other by three amide linker (—C(O)NH—) groups, where each        of the polyoxyalkylene groups has a molecular weight of about        750 Da, such that the polymer moiety has molecular weight of        about 3000 Da; or    -   (c) consist of five polyoxyethylene groups covalently bound to        each other by four amide linker (—C(O)NH—) groups, where the        five polyoxyalkylene groups have molecular weights of about 500,        1000, 250, 1000, and 250 Da, respectively.

These polymer moieties are included only as examples; those skilled inthe art will recognize other polymer moieties that can be suitablyemployed in the compounds of the invention.

Non-limiting examples of reagents useful for making the polymer moietiesof the invention include the following:

HO(alkylene-O)_(pp)R^(bb) mono-capped mono-hydroxy PEG (mPEG)H₂N(alkylene-O)_(pp)R^(bb) mono-capped mono-amino PEGHO(alkylene-O)_(pp)R—OH non-capped di-hydroxy PEGH₂N(alkylene-O)_(pp)R—OH non-capped mono-amino PEGwhere pp and alkylene are as defined herein and R^(bb) is preferablyselected from the group consisting of alkyl and substituted alkyl.

Specific examples of such reagents include:

In some embodiments, the particles are made by providing an aqueoussolution in a first reservoir and an organic lipid solution (i.e., asolution of a compound of the invention in water) in a second reservoirand mixing the aqueous solution with the organic lipid solution so as tosubstantially instantaneously produce a liposome encapsulating, e.g., aninterfering RNA. In some embodiments, the particles are made byformation of hydrophobic intermediate complexes in eitherdetergent-based or organic solvent-based systems, followed by removal ofthe detergent or organic solvent. Preferred embodiments arecharge-neutralized.

In one embodiment, the interfering RNA is transcribed from a plasmid andthe plasmid is combined with cationic lipids in a detergent solution toprovide a coated nucleic acid-lipid complex. The complex is thencontacted with non-cationic lipids to provide a solution of detergent, anucleic acid-lipid complex and non-cationic lipids, and the detergent isthen removed to provide a solution of serum-stable nucleic acid-lipidparticles, in which the plasmid comprising an interfering RNA templateis encapsulated in a lipid bilayer. The particles thus formed have asize of about 50-500 nm.

In another embodiment, stable lipid particles are formed by preparing amixture of cationic lipids and non-cationic lipids in an organicsolvent; contacting an aqueous solution of nucleic acids comprising,e.g., interfering RNA with the mixture of cationic and non-cationiclipids to provide a clear single phase; and removing the organic solventto provide a suspension of nucleic acid-lipid particles, in which thenucleic acid is encapsulated in a lipid bilayer, and the particles arestable in serum and have a size of about 50-500 nm.

Particles and complexes of the invention, e.g., complexes of a compoundof any of formula I-VI with a siRNA, having desired sizes and/or chargecan be obtained by passing mixtures prepared as above through suitablefilters. See, e.g., Examples 32 and 33 below.

The lipid particles of the invention are useful for the therapeuticdelivery of nucleic acids comprising a siRNA sequence. In particular, itis an object of this invention to provide in vitro and in vivo methodsfor treatment of a disease in a mammal by downregulating or silencingthe translation of a target nucleic acid sequence. In these methods, asiRNA molecule is formulated into a nucleic acid-lipid particle, and theparticles are administered to patients requiring such treatment (e.g., apatient diagnosed with a disease or disorder associated with theexpression or overexpression of a gene comprising the target nucleicacid sequence). Alternatively, cells are removed from a patient, thesiRNA is delivered in vitro, and the cells are reinjected into thepatient. In one embodiment, the present invention provides for a methodof introducing a siRNA molecule into a cell by contacting a cell with anucleic acid-lipid particle comprising of a cationic lipid, anon-cationic lipid, a conjugated lipid that inhibits aggregation, and asiRNA. In another embodiment, the present invention provides for amethod of introducing a siRNA molecule into a cell by contacting a cellwith a nucleic acid-lipid particle comprising of a cationic lipid, aconjugated lipid that inhibits aggregation, and a siRNA. In yet anotherembodiment, the present invention provides for a method of introducing asiRNA molecule into a cell by contacting a cell with a nucleicacid-lipid particle comprising of a cationic lipid, and a siRNA.

The lipid particle may be administered, e.g., intravenously, orintraperitoneally. In one embodiment, at least about 10% of the totaladministered dose of the nucleic acid-lipid particles is present inplasma about 1, 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours afterinjection. In other embodiments, more than 20%, 30%, 40% and as much as60%, 70% or 80% of the total injected dose of the nucleic acid-lipidparticles is present in plasma 1, 6, 12, 24, 36, 48, 60, 72, 84, or 96hours after injection. In one embodiment, the presence of a siRNA incells in a target tissue (i.e., lung, liver, tumor, vascular endotheliumor at a site of inflammation) is detectable at 24, 48, 72 and 96 hoursafter administration. In one embodiment, downregulation of expression ofthe target sequence is detectable at 24, 48, 72 and 96 hours afteradministration. In one embodiment, downregulation of expression of thetarget sequence occurs preferentially in tumor cells or in cells at asite of inflammation or any other disease tissue. In one embodiment, thepresence of a siRNA in cells at a site distal to the site ofadministration is detectable at least four days after intravenousinjection of the nucleic acid-lipid particle. In another embodiment, thepresence of a siRNA in of cells in a target tissue (i.e., lung, liver,tumor or at a site of inflammation) is detectable at least four daysafter injection of the nucleic acid-lipid particle.

The particles are suitable for use in intravenous nucleic acid transferas they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites andtarget cell populations. The invention also provides forpharmaceutically acceptable compositions comprising a lipid particle.

The particles are suitable for use in intravenous nucleic acid transferas they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites andtarget cell populations.

The stable nucleic acid-lipid particles described herein typicallycomprise a nucleic acid (e.g., a siRNA sequence or a DNA sequenceencoding a siRNA sequence), a cationic lipid, a noncationic lipid and abilayer stabilizing component such as, e.g., a conjugated lipid thatinhibits aggregation of the lipid particles. The lipid particles of thepresent invention have a mean diameter of less than about 500 nm and aresubstantially nontoxic. In addition, nucleic acids encapsulated in thelipid particles of the present invention are resistant in aqueoussolution to degradation with a nuclease.

The nucleic acid component of the nucleic acid-lipid particles typicallycomprise an interfering RNA (i.e., siRNA), which can be provided inseveral forms including, e.g. as one or more isolated small-interferingRNA (siRNA) duplexes, longer double-stranded RNA (dsRNA) or as siRNA ordsRNA transcribed from a transcriptional cassette in a DNA plasmid.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected etc.), or can represent a single target sequence.RNA can be naturally occurring, e.g., isolated from tissue or cellsamples, synthesized in vitro, e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA; or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a ds RNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly encapsulated inthe nucleic acid-lipid particles or can be digested in vitro prior toencapsulation.

Alternatively, one or more DNA plasmids encoding one or more siRNAtemplates are encapsulated in a nucleic acid-lipid particle. siRNA canbe transcribed as sequences that automatically fold into duplexes withhairpin loops from DNA templates in plasmids having RNA polymerase IIItranscriptional units, for example, based on the naturally occurringtranscription units for small nuclear RNA U6 or human RNase P RNA H1(see, Brummelkamp, et al., Science 296:550 (2002); Donze, et al.,Nucleic Acids Res. 30:e46 (2002); Paddison, et al., Genes Dev. 16:948(2002); Yu, et al., Proc. Natl. Acad. Sci. 99:6047 (2002); Lee, et al.,Nat. Biotech. 20:500 (2002); Miyagishi, et al., Nat. Biotech. 20:497(2002); Paul, et al., Nat. Biotech. 20:505 (2002); and Sui, et al.,Proc. Natl. Acad. Sci. 99:5515 (2002)). Typically, a transcriptionalunit or cassette will contain an RNA transcript promoter sequence, suchas an H1-RNA or a U6 promoter, operably linked to a template fortranscription of a desired siRNA sequence and a termination sequence,comprised of 2-3 uridine residues and a polythymidine (T5) sequence(polyadenylation signal) (Brummelkamp, Science, supra). The selectedpromoter can provide for constitutive or inducible transcription.Compositions and methods for DNA-directed transcription of RNAinterference molecules is described in detail in U.S. Pat. No.6,573,099, incorporated herein by reference. Preferably, the synthesizedor transcribed siRNA have 3′ overhangs of about 1-4 nucleotides,preferably of about 2-3 nucleotides and 5′ phosphate termini (Elbashir,et al., Genes Dev. 15:188 (2001); Nykanen, et al., Cell 107:309 (2001)).The transcriptional unit is incorporated into a plasmid or DNA vectorfrom which the interfering RNA is transcribed. Plasmids suitable for invivo delivery of genetic material for therapeutic purposes are describedin detail in U.S. Pat. Nos. 5,962,428 and 5,910,488, both of which areincorporated herein by reference. The selected plasmid can provide fortransient or stable delivery of a target cell. It will be apparent tothose of skill in the art that plasmids originally designed to expressdesired gene sequences can be modified to contain a transcriptional unitcassette for transcription of siRNA.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994)).

Generally, it is desired to deliver the nucleic acid-lipid particles todownregulate or silence the translation (i.e., expression) of a geneproduct of interest. Suitable classes of gene products include, but arenot limited to, genes associated with viral infection and survival,genes associated with metabolic diseases and disorders (e.g., diseasesand disorders in which the liver is the target, and liver diseases anddisorders) and disorders, genes associated with tumorigenesis and celltransformation, angiogenic genes, immunomodulator genes, such as thoseassociated with inflammatory and autoimmune responses, ligand receptorgenes, and genes associated with neurodegenerative disorders.

Genes associated with viral infection and survival include thoseexpressed by a virus in order to bind, enter and replicate in a cell. Ofparticular interest are viral sequences associated with chronic viraldiseases. Viral sequences of particular interest include sequences ofHepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota,et al., EMBO Rep. 4:602 (2003); Schlomai, et al., Hepatology 37:764(2003); Wilson, et al., Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia,et al., Proc. Natl. Acad. Sci. 100:2014 (2003); and FIELDS VIROLOGY(Knipe et al. eds. 2001)), Human Immunodeficiency Virus (HIV) (Banerjea,et al., Mol. Ther. 8:62 (2003); Song, et al., J. Virol. 77:7174 (2003);Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad. Sci.100:183 (2003)), Herpes viruses (Jia, et al., J. Viral. 77:3301 (2003)),and Human Papilloma Viruses (HPV) (Hall, et al., J. Viral. 77:6066(2003); Jiang, et al., Oncogene 21:6041 (2002)). Examplary hepatitisviral nucleic acid sequences that can be silenced include but are notlimited to: nucleic acid sequences involved in transcription andtranslation (e.g., En1, En2, X, P), nucleic acid sequences encodingstructural proteins (e.g., core proteins including C and C-relatedproteins; capsid and envelope proteins including S, M, and/or Lproteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001,supra). Exemplary Hepatitis C nucleic acid sequences that can besilenced include but are not limited to: serine proteases (e.g.,NS3/NS4), helicases (e.g. NS3), polymerases (e.g., NS5B), and envelopeproteins (e.g., E1, E2, and p7). Hepatitis A nucleic acid sequences areset forth in e.g., Genbank Accession No. NC_(—)001489; Hepatitis Bnucleic acid sequences are set forth in, e.g., Genbank Accession No.NC_(—)003977; Hepatitis C nucleic acid sequences are set forth in, e.g.,Genbank Accession No. NC_(—)004102; Hepatitis D nucleic acid sequenceare set forth in, e.g., Genbank Accession No. NC_(—)001653; Hepatitis Enucleic acid sequences are set forth in e.g., Genbank Accession No.NC_(—)001434; and Hepatitis G nucleic acid sequences are set forth ine.g., Genbank Accession No. NC_(—)001710. Silencing of sequences thatencode genes associated with viral infection and survival canconveniently be used in combination with the administration ofconventional agents used to treat the viral condition.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, for example genes expressed in, for example, dyslipidemia(e.g., liver X receptors (e.g., LXRα and LXRβ, Genback Accession No.NM_(—)007121), farnesoid X receptors (FXR) (Genback Accession No.NM_(—)005123), sterol-regulatory element binding protein (SREBP), Site-1protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGcoenzyme-A reductase), Apolipoprotein (ApoB), and Apolipoprotein (ApoE))and diabetes (e.g., Glucose 6-phosphatase) (see, e.g., Forman et al.,Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995), Zavackiet al., PNAS USA 94:7909 (1997); Sakai, et al., Cell 85:1037-1046(1996); Duncan, et al., J. Biol. Chem. 272:12778-12785 (1997); Willy, etal., Genes Dev. 9(9):1033-45 (1995); Lehmann, et al., J. Biol. Chem.272(6):3137-3140 (1997); Janowski, et al., Nature 383:728-731 (1996);Peet, et al., Cell 93:693-704 (1998)). One of skill in the art willappreciate that genes associated with metabolic diseases and disorders(e.g., diseases and disorders in which the liver is a target and liverdiseases and disorders) include genes that are expressed in the liveritself as well as and genes expressed in other organs and tissues.Silencing of sequences that encode genes associated with metabolicdiseases and disorders can conveniently be used in combination with theadministration of conventional agents used to treat the disease ordisorder.

Examples of gene sequences associated with tumorigenesis and celltransformation include translocation sequences such as MLL fusion genes,BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al., Blood101: 1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO andAML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressedsequences such as multidrug resistance genes (Nieth, et al., FEBS Lett.545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cyclins (Li, etal., Cancer Res. 63:3593 (2003); Zou, et al., Genes Dev. 16:2923(2002)), beta-Catenin (Verma, et al., Clin Cancer Res. 9:1291 (2003)),telomerase genes (Kosciolek, et al., Mol Cancer Ther. 2:209 (2003)),c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp. Cell Res. 285:39(2003)); and mutated sequences such as RAS (reviewed in Tuschl andBorkhardt, Mol. Interventions, 2:158 (2002)). Silencing of sequencesthat encode DNA repair enzymes find use in combination with theadministration of chemotherapeutic agents (Collis, et al., Cancer Res.63:1550 (2003)). Genes encoding proteins associated with tumor migrationare also target sequences of interest, for example, integrins, selectinsand metalloproteinases. The foregoing examples are not exclusive. Anywhole or partial gene sequence that facilitates or promotestumorigenesis or cell transformation, tumor growth or tumor migrationcan be included as a template sequence

Angiogenic genes are able to promote the formation of new vessels. Ofparticular interest is Vascular Endothelial Growth Factor (VEGF) (Reich,et al., Mol. Vis. 9:210 (2003)).

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include cytokines such asgrowth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF,GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill, et al.,J. Immunol. 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons(e.g., IFN-α, IFN-β, IFN-γ, etc.) and TNF. Fas and Fas Ligand genes arealso immunomodulator target sequences of Interest (Song, et al., Nat.Med. 9:347 (2003)). Genes encoding secondary signaling molecules inhematopoietic and lymphoid cells are also included in the presentinvention, for example, Tec family kinases, such as Bruton's tyrosinekinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 (2002)).

Cell receptor ligands of the invention can be proteins or steroidmolecules. Cell receptor ligands include ligands that are able to bindto cell surface receptors (e.g., insulin receptor, EPO receptor,G-protein coupled receptors, receptors with tyrosine kinase activity,cytokine receptors, growth factor receptors, etc.), to modulate (e.g.,inhibit, activate, etc.) the physiological pathway that the receptor isinvolved in (e.g., glucose level modulation, blood cell development,mitogenesis, etc.). Examples of cell surface receptor ligands includecytokines, growth factors, interleukins, interferons, erythropoietin(EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.).Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats), find use in silencing pathogenic sequences inneurodegenerative disorders caused by the expansion of trinucleotiderepeats, such as spinobulbular muscular atrophy and Huntington's Disease(Caplen, et al., Hum. Mol. Genet. 11:175 (2002)). Cell receptor ligandsalso include ligands that do not bind to cell surface receptors but tointracellular receptors (e.g., steroid receptors located in the nucleus,and cytoplasm, inositol phosphate receptors located in the endoplasmicreticulum). Examples of intracellular receptor ligands includelipophilic hormones like steroid hormones, inositol trisphosphate, andintracrine peptide hormones.

DEFINITIONS

As used herein, the term “alkyl” includes those alkyl groups containingfrom 1 to 10 carbon atoms. Alkyl groups may be straight, or branched.Examples of “alkyl” include methyl, ethyl, propyl, isopropyl, butyl,iso-, sec- and tert-butyl, pentyl, isopentyl, hexyl, 3-methylhexyl,heptyl, octyl, nonyl, 3-ethylbutyl, and the like. A preferred alkylgroup is C₁-C₆ alkyl. The alkyl groups of the invention may beoptionally substituted with various groups as provided herein. Thus, anycarbon atom available for substitution may be further bonded to avariety of substituents, such as, for example, halogen, OH, NO₂, CN,NH₂, C₁-C₈ alkyl, C₁-C₈ alkoxy, NH(C₁-C₈ alkyl). N(C₁-C₈ alkyl) (C₁-C₈alkyl), C₃-C₁₀ cycloalkyl, (C₃-C₁₀ cycloalkyl)alkyl, (C₃-C₁₀cycloalkyl)alkoxy, C₂-C₉ heterocycloalkyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,halo(C₁-C₈)alkyl, halo(C₁-C₈)alkoxy, oxo, amino (C₁-C₈)alkyl, mono- anddi(C₁-C₈ alkyl)amino(C₁-C₈)alkyl, C₁-C₈ acyl, C₁-C₈ acyloxy, C₁-C₈sulfonyl, C₁-C₈ thio, C₁-C₈ sulfonamido, and C₁-C₈ aminosulfonyl.

The term “alkylene” refers to divalent saturated aliphatic hydrocarbylgroups preferably having from 1 to 5 and more preferably 1 to 3 carbonatoms which are either straight-chained or branched. This term isexemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),n-propylene (—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—) and the like.

The term “alkyleneoxy” refers to divalent saturated aliphatichydrocarbyl groups bound to an oxygen, where the aliphatic hydrocarbylgroups preferably have from 1 to 5 and more preferably 1 to 3 carbonatoms which are either straight-chained or branched.

The term “aryl” refers to an aromatic hydrocarbon ring system containingat least one aromatic ring. The aromatic ring may optionally be fused orotherwise attached to other aromatic hydrocarbon rings or non-aromatichydrocarbon rings. Examples of aryl groups include, for example, phenyl,naphthyl, anthracenyl 1,2,3,4-tetrahydronaphthalene, indenyl,2,3-dihydroindenyl, and biphenyl. Preferred examples of aryl groupsinclude phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene, and2,3-dihydroindenyl. More preferred aryl groups are phenyl and naphthyl.Most preferred is phenyl. The aryl groups of the invention may beoptionally substituted with various groups as provided herein. Thus, anycarbon atom present within an aryl ring system and available forsubstitution may be further bonded to a variety of ring substituents,such as, for example, halogen, OH, NO₂, CN, NH₂, C₁-C₈ alkyl, C₁-C₈alkoxy, NH(C₁-C₈ alkyl), N(C₁-C₈ alkyl) (C₁-C₈ alkyl), C₃-C₁₀cycloalkyl, (C₃-C₁₀ cycloalkyl)alkyl, (C₃-C₁₀ cycloalkyl)alkoxy, C₂-C₉heterocycloalkyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halo(C₁-C₈)alkyl,halo(C₁-C₈) alkoxy, oxo, amino(C₁-C₈)alkyl, mono- and di(C₁-C₈alkyl)amino(C₁-C₈) alkyl, C₁-C₈ acyl, C₁-C₈ acyloxy, C₁-C₈ sulfonyl,C₂-C₈ thio, C₁-C₈ sulfonamido, and C₁-C₈ aminosulfonyl.

The term “cycloalkyl” refers to a C₃-C₈ cyclic hydrocarbon. Examples ofcycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl. More preferred are C₃-C₆ cycloalkyl groups.The cycloalkyl groups of the invention may be optionally substitutedwith various groups as provided herein. Thus, any carbon atom presentwithin a cycloalkyl ring system and available for substitution may befurther bonded to a variety of ring substituents, such as, for example,halogen, OH, NO₂, CN, NH, C₁-C₈ alkyl, C₁-C₈ alkoxy, NH(C₁-C₃ alkyl),N(C₁-C₈ alkyl) (C₁-C₈ alkyl), C₃-C₁₀ cycloalkyl, (C₃-C₁₀cycloalkyl)alkyl, (C₃-C₁₀ cycloalkyl)alkoxy, C₂-C₉ heterocycloalkyl,C₁-C₈ alkenyl, C₁-C₈ alkynyl, halo(C₁-C₈)alkyl, halo(C₁-C₃)alkoxy, oxo,amino(C₁-C₃)alkyl, mono- and di(C₁-C₈alkyl)amino(C₁-C₈)alkyl.

The term “heterocycloalkyl” refers to a ring or ring system containingat least one heteroatom selected from nitrogen, oxygen, and sulfur,wherein said heteroatom is in a non-aromatic ring and the ring system isattached to the parent group by a member of (one of) the non-aromaticring(s). The heterocycloalkyl ring is optionally fused to otherheterocycloalkyl rings and/or non-aromatic hydrocarbon rings, and/orphenyl rings. Thus, heterocycloalkyl groups suitable for the inventionhave at least 3 members, and may have up to 20 members. Preferredheterocycloalkyl groups have from 3 to members. Certain more preferredheterocycloalkyl groups have from 8-10 members. Other more preferredheterocycloalkyl groups have 5 or 6 members. Examples ofheterocycloalkyl groups include, for example,1,2,3,4-tetrahydroisoquinolinyl, 1,2-dihydroquinolinyl,1,2,3,4-tetrahydroquinolinyl, benzo[1,4]oxazinyl,2,3-dihydrobenzo[1,4]oxazinyl, indolinyl, benzo[1,3]dioxolyl,2H-chromenyl, piperazinyl, morpholinyl, piperidinyl, piperazinyl,tetrahydrofuranyl, pyrrolidinyl, pyridinonyl, azetidinyl, aziridinyl,and pyrazolidinyl. Preferred heterocycloalkyl groups includepiperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, pyridinonyl,dihydropyrrolidinyl, azetidinyl, aziridinyl, 2,3,4-tetrahydroquinolinyl,2,3-dihydrobenzo[1,4]oxazinyl, indolinyl, benzo[1,3]dioxolyl, andpyrrolidinonyl. More preferred heterocycloalkyl groups are pyrrolidinly,piperidinyl, azetidinyl, aziridinyl, piperazinyl, morpholinyl,1,2,3,4-tetrahydroquinolinyl, 2,3-dihydrobenzo[1,4]oxazinyl, indolinyl,and benzo[1,3]dioxolyl. The heterocycloalkyl groups of the invention maybe optionally substituted with various groups as provided herein. Thus,any atom present within a heterocycloalkyl ring and available forsubstitution may be further bonded to a variety of ring substituents,such as, for example, halogen, OH, NO₂, CN, NH₂, C₁-C₈ alkyl, C₁-C₈alkoxy, NH(C₁-C₈ alkyl), N(C₁-C₈ alkyl) (C₁-C₈ alkyl), C₃-C₁₀cycloalkyl, (C₃-C₁₀ cycloalkyl)alkyl, (C₃-C₁₀ cycloalkyl)alkoxy, C₂-C₉heterocycloalkyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halo(C₁-C₈)alkyl,halo(C₁-C₈)alkoxy, oxo, amino(C₁-C₈)alkyl and mono- and di(C₁-C₈alkyl)amino(C₁-C₈)alkyl.

The term “heteroaryl” refers to an aromatic ring system containing atleast one heteroatom selected from nitrogen, oxygen, and sulfur and thering system is attached to the parent group by a member of (one of) thearomatic ring(s). The heteroaryl ring may be fused to one or moreheteroaryl rings, aromatic or non-aromatic hydrocarbon rings orheterocycloalkyl rings. Thus, heteroaryl groups suitable for theinvention have at least 5 members, and may have up to 20 members.Examples of heteroaryl groups include, for example, pyridine, furan,thienyl, 5,6,7,8-tetrahydroisoquinolinyl and pyrimidinyl. Preferredheteroaryl groups include thienyl, benzothienyl, pyridyl, quinolinyl,pyrazolyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl,dibenzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl,isothiazolyl, benzisothiazolyl, triazolyl, pyrrolyl, indolyl,5,6-dihydroquinazolinyl, 4,5,6,7-tetrahydroindolyl,4,5-dihydro-2H-indazolyl, 5,6-dihydroquinolinyl, pyrazolyl, andbenzopyrazolyl. More preferred heteroaryl groups are benzothiazolyl,pyridyl, pyrazolyl, and quinolinyl. The heteroaryl groups of theinvention may be optionally substituted with various groups as providedherein. Thus, any carbon atom present within an heteroaryl ring systemand available for substitution may be further bonded to a variety ofring substituents, such as, for example, halogen, OH, NO₂, CN, NH₂,C₁-C₈ alkyl, C₁-C₈ alkoxy, NH(C₁-C₈ alkyl), N(C₁-C₈ alkyl) (C₁-C₈alkyl), C₃-C₁₀ cycloalkyl, (C₃-C₁₀ cycloalkyl)alkyl, (C₃-C₁₀cycloalkyl)alkoxy, C₂-C₉ heterocycloalkyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,halo(C₁-C₈alkyl, halo(C₁-C₈)alkoxy, oxo, amino(C₁-C₈)alkyl and mono- anddi(C₁-C₈ alkyl)amino(C₁-C₈)alkyl.

By “anti-proliferative activity” as used herein is meant biologicalactivity against any disease, condition, trait, genotype or phenotypecharacterized by unregulated cell growth or replication as is known inthe art; including leukemias, for example, acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia(ALL), and chronic lymphocytic leukemia, AIDS related cancers such asKaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

The term “aptamer” means a nucleic acid that binds to another molecule.This binding interaction does not encompass standard nucleicacid/nucleic acid hydrogen bond formation exemplified by Watson-Crickbasepair formation (e.g., A binds to U or T and G binds to C), butencompasses all other types of non-covalent (or in some cases covalent)binding. Non-limiting examples of non-covalent binding include hydrogenbond formation, electrostatic interaction, Van der Waals interaction andhydrophobic interaction. An aptamer may bind to another molecule by anyor all of these types of interaction, or in some cases by covalentinteraction. Covalent binding of an aptamer to another molecule mayoccur where the aptamer or target molecule contains a chemicallyreactive or photoreactive moiety. The term “aptamer” refers to a nucleicacid that is capable of forming a complex with an intended targetsubstance. “Target-specific” means that the aptamer binds to a targetanalyte with a much higher degree of affinity than it binds tocontaminating materials.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules include antibodies (e.g., monoclonal, chimeric,humanized etc.), cholesterol, hormones, antivirals, peptides, proteins,chemotherapeutics, small molecules, vitamins, co-factors, nucleosides,nucleotides, oligonucleotides, enzymatic nucleic acids (e.g., ribozymes,etc.), antisense nucleic acids, triplex forming oligonucleotides, 2,5-Achimeras, dsRNA, (e.g., siNA, siRNA, etc.), allozymes, aptamers, decoys,ribosomal RNA, antisense polynucleotides of RNA or DNA or combinationsof RNA and DNA, miRNA, shRNA, and polynucleotides of genomic DNA, cDNA,or mRNA that encode for a therapeutically useful protein, and analogsthereof. Biologically active molecules of the invention also includemolecules capable of modulating the pharmacokinetics and/orpharmacodynamics of other biologically active molecules, for example,lipids and polymers such as polyamines, polyamides, polyethylene glycoland other polyethers. In certain embodiments, the term biologicallyactive molecule is used interchangeably with the term “molecule” or“molecule of interest” herein.

By “cationic lipid” as used herein is meant any lipophilic compoundhaving a net positive charge at about physiological pH, such as acompound having any of Formulae I-VI.

By “neutral lipid” as used herein is meant any lipophilic compound otherthan a cationic lipid as defined herein that does not bear a net chargeat about physiological pH. Suitable compounds having no net charge atabout physiological pH include zwitterions.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The term “double stranded RNA” or “dsRNA” as used herein refers to adouble stranded RNA molecule capable of RNA interference, includingshort interfering RNA (siRNA).

By “gene” is meant a nucleic acid that encodes RNA, for example, nucleicacid sequences including, but not limited to, structural genes encodinga polypeptide. A gene or target gene can also encode a functional RNA(fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA),micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA(smRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA(tRNA) and precursor RNAs thereof.

By “inhibit” or “inhibition”, it is meant that the expression of thegene, or level of RNA molecules or equivalent RNA molecules encoding oneor more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is reduced below that observed in theabsence of the nucleic acid molecules and the molecules of theinvention. In one embodiment, inhibition with a siNA molecule is belowthat level observed in the presence of an inactive or attenuatedmolecule. In another embodiment, inhibition with siNA molecules is belowthat level observed in the presence of, for example, a siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition of gene expression with a nucleic acid molecule of theinstant invention is greater in the presence of the nucleic acidmolecule than in its absence. In one embodiment, inhibition of geneexpression is associated with post transcriptional silencing, such asRNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) orinhibition of translation. In one embodiment, inhibition is associatedwith pretranscriptional silencing.

The terms “linker” and “linker group” refer to polyvalent, preferablydivalent, moieties that connect one or more molecules to each other.Examples are moieties that connect portions of a polymer moiety Gtogether or connect a targeting ligand T to a polymer moiety G. Linkerscan be biodegradable or biostable. Biodegradable linkers are designedsuch that their stability can be modulated for a particular purpose,such as delivery to a particular tissue or cell type. Examples ofsuitable linker groups include the following groups: —C(O)—, —O—,—O—C(O)O—, —C(O)CH₂CH₂C(O)—, —S—S—, —NR³—, —NR³C(O)O—, —OC(O)NR³—,—NR³C(O)—C(O)NR³—, —NR³C(O)NR³—, -alkylene-NR³C(O)O—,-alkylene-NR³C(O)NR³—, -alkylene-OC(O) NR³—, -alkylene-NR³—,-alkylene-O—, -alkylene-NR³C(O)-alkylene-C(O)NR³—, —NR³C(O)O-alkylene-,—NR³C(O)NR³-alkylene-, —OC(O) NR³-alkylene, —NR³-alkylene-,—O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,-alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR³-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, and -alkyleneoxy-NR³C(O)O-alkyleneoxy-, where R³is hydrogen, or optionally substituted alkyl, and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—,-alkylene-OC(O)NR³-alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—,alkylene-C(O)NR³—, —NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-,—OC(O)NR³-alkylene-, —NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O-alkylene-,-alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O)NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-,-alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is asdefined above.

The term “targeting ligand” refers to any compound or molecule, such asa drug, peptide, hormone, or neurotransmitter that is capable ofinteracting with another compound, such as a receptor, either directlyor indirectly. The receptor that interacts with a ligand can be presenton the surface of a cell or can alternately be an intercellularreceptor. Interaction of the ligand with the receptor can result in abiochemical reaction, or can simply be a physical interaction orassociation. Non-limiting examples of ligands include pharmacologicallyactive small molecules, endosomolytic agents, fusogenic peptides, cellmembrane permeating agents, charge masking agents, and nucleic acids.Other non-limiting examples of ligands include sugars and carbohydratessuch as galactose, galactosamine, and N-acetyl galactosamine; hormonessuch as estrogen, testosterone, progesterone, glucocortisone,adrenaline, insulin, glucagon, cortisol, vitamin D, thyroid hormone,retinoic acid, and growth hormones; growth factors such as VEGF, EGF,NGF, and PDGF; cholesterol; bile acids; neurotransmitters such as GABA,Glutamate, acetylcholine; NOGO; inostitol triphosphate; diacylglycerol;epinephrine; norepinephrine; peptides, vitamins such as folate,pyridoxine, and biotin, drugs such as acetylsalicylic acid and naproxen,antibodies and any other molecule that can interact with a receptor invivo or in vitro. The ligand can be attached to a compound of theinvention using a linker molecule, such as an amide, carbonyl, ester,peptide, disulphide, silane, nucleoside, abasic nucleoside, polyether,polyamine, polyamide, carbohydrate, lipid, polyhydrocarbon, phosphateester, phosphoramidate, thiophosphate, alkylphosphate, or photolabilelinker. In one embodiment, the linker is a biodegradable linker.Conditions suitable for cleavage can include but are not limited to pH,UV irradiation, enzymatic activity, temperature, hydrolysis,elimination, and substitution reactions, and thermodynamic properties ofthe linkage.

The term “lipid” as used herein, refers to any lipophilic compound.Non-limiting examples of lipid compounds include fatty acids and theirderivatives, including straight chain, branched chain, saturated andunsaturated fatty acids, carotenoids, terpenes, bile acids, andsteroids, including cholesterol and derivatives or analogs thereof.

The term “lipid particle” or “lipid particle composition” refers to acomposition comprising one or more biologically active moleculesindependently or in combination with a cationic lipid, a neutral lipid,and/or a polyethyleneglycol-diacylglycerol conjugate (i.e.,polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB). A formulated molecular composition can further comprisecholesterol or a cholesterol derivative. The cationic lipid of theinvention can comprise a compound having any of Formulae I-VI ,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane(CpLin DMA), N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or amixture thereof. The neutral lipid can comprisedioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. The PEG conjugate can comprise a PEG-dilaurylglycerol(C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16),a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16),PEG-disterylglycamide (C18), PEG-cholesterol, or PEG-DMB. The cationiclipid component can comprise from about 2% to about 60%, from about 5%to about 45%, from about 5% to about 15%, or from about 40% to about 50%of the total lipid present in the formulation. The neutral lipidcomponent can comprise from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation. The PEG-DAGconjugate (e.g., polyethyleneglycol diacylglycerol (PEG-DAG),PEG-cholesterol, or PEG-DMB) can comprise from about 1% to about 20%, orfrom about 4% to about 15% of the total lipid present in theformulation. The cholesterol component can comprise from about 10% toabout 60%, or from about 20% to about 45% of the total lipid present inthe formulation. In one embodiment, a formulated molecular compositionof the invention comprises a cationic lipid component comprising about7.5% of the total lipid present in the formulation, a neutral lipidcomprising about 82.5% of the total lipid present in the formulation,and a PEG conjugate comprising about 10% of the total lipid present inthe formulation. In one embodiment, a formulated molecular compositionof the invention comprises a biologically active molecule, DODMA, DSPC,and a PEG-DAG conjugate. In one embodiment, the PEG-DAG conjugate isPEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In anotherembodiment, the formulated molecular composition also comprisescholesterol or a cholesterol derivative.

By “nanoparticle” is meant a microscopic particle whose size is measuredin nanometers. Nanoparticles of the invention typically range from about1 to about 999 nm in diameter, and can include an encapsulated orenclosed biologically active molecule.

By “PEG” is meant, any polyethylene glycol or other polyalkylene etheror equivalent polymer.

The terms “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, and “short interfering nucleic acid molecule” as usedherein refer to any nucleic acid molecule capable of inhibiting or downregulating gene expression or viral replication by mediating RNAinterference “RNAi” or gene silencing in a sequence-specific manner. Forexample the siNA can be a double-stranded nucleic acid moleculecomprising self-complementary sense and antisense regions, wherein theantisense region comprises nucleotide sequence that is complementary tonucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof. The siNA can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e., each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure, for examplewherein the double stranded region is about 15 to about 30, e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 basepairs; the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof(e.g., about 15 to about 25 or more nucleotides of the siNA molecule arecomplementary to the target nucleic acid or a portion thereof).Alternatively, the siNA is assembled from a single oligonucleotide,where the self-complementary sense and antisense regions of the siNA arelinked by means of a nucleic acid based or non-nucleic acid-basedlinker(s). The siNA can be a polynucleotide with a duplex, asymmetricduplex, hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a separate target nucleic acid molecule or a portion thereofand the sense region having nucleotide sequence corresponding to thetarget nucleic acid sequence or a portion thereof. The siNA can be acircular single-stranded polynucleotide having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises nucleotide sequence thatis complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active siNA molecule capable ofmediating RNAi. The siNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (forexample, where such siNA molecule does not require the presence withinthe siNA molecule of nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell., 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or5′,3′-diphosphate. In certain embodiments, the siNA molecule of theinvention comprises separate sense and antisense sequences or regions,wherein the sense and antisense regions are covalently linked bynucleotide or non-nucleotide linkers molecules as is known in the art,or are alternately non-covalently linked by ionic interactions, hydrogenbonding, van der waals interactions, hydrophobic interactions, and/orstacking interactions. In certain embodiments, the siNA molecules of theinvention comprise nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, the siNAmolecule of the invention interacts with nucleotide sequence of a targetgene in a manner that causes inhibition of expression of the targetgene. As used herein, siNA molecules need not be limited to thosemolecules containing only RNA, but further encompasseschemically-modified nucleotides and non-nucleotides. In certainembodiments, the short interfering nucleic acid molecules of theinvention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicantdescribes in certain embodiments short interfering nucleic acids that donot require the presence of nucleotides having a 2′-hydroxy group formediating RNAi and as such, short interfering nucleic acid molecules ofthe invention optionally do not include any ribonucleotides (e.g.,nucleotides having a 2′-OH group). Such siNA molecules that do notrequire the presence of ribonucleotides within the siNA molecule tosupport RNAi can however have an attached linker or linkers or otherattached or associated groups, moieties, or chains containing one ormore nucleotides with 2′-OH groups. Optionally, siNA molecules cancomprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of thenucleotide positions. The modified short interfering nucleic acidmolecules of the invention can also be referred to as short interferingmodified oligonucleotides. As used herein, the term siNA is meant to beequivalent to other terms used to describe nucleic acid molecules thatare capable of mediating sequence specific RNAi, for example shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),short hairpin RNA (shRNA), short interfering oligonucleotide, shortinterfering nucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. Non limiting examples of siNA molecules of theinvention are shown in U.S. Ser. No. 11/234,730, filed Sep. 23, 2005,incorporated by reference in its entirety herein. Such siNA moleculesare distinct from other nucleic acid technologies known in the art thatmediate inhibition of gene expression, such as ribozymes, antisense,triplex forming, aptamer, 2,5-A chimera, or decoy oligonucleotides.

By “target” as used herein is meant, any target protein, peptide, orpolypeptide encoded by a target gene. The term “target” also refers tonucleic acid sequences encoding any target protein, peptide, orpolypeptide having target activity, such as encoded by target RNA. Theterm “target” is also meant to include other target encoding sequence,such as other target isoforms, mutant target genes, splice variants oftarget genes, and target gene polymorphisms.

Pharmaceutical Compositions

The compounds of the invention may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. In addition, there is provided a pharmaceuticalformulation comprising a compound of the invention and apharmaceutically acceptable carrier. One or more compounds of theinvention may be present in association with one or more non-toxicpharmaceutically acceptable carriers and/or diluents and/or adjuvants,and if desired other active ingredients. The pharmaceutical compositionscontaining compounds of the invention may be in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to anymethod known in the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preservative agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques. In some cases such coatings may be prepared by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatincapsules, wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

Formulations for oral use may also be presented as lozenges.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide palatable oral preparations. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations may also contain a demulcent, preservative and flavoringand coloring agent. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds of the invention may also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Compounds of the invention may be administered parenterally in a sterilemedium. The drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anesthetics, preservatives and buffering agentscan be dissolved in the vehicle.

For disorders of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical gel, spray,ointment or cream, or as a suppository, containing the activeingredients in a total amount of, for example, 0.075 to 30% w/w,preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither paraffinic or a water-miscible ointment base.

Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base. If desired, the aqueous phase of the creambase may include, for example at least 30% w/w of a polyhydric alcoholsuch as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol,polyethylene glycol and mixtures thereof. The topical formulation maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethylsulfoxide andrelated analogs. The compounds of this invention can also beadministered by a transdermal device. Preferably topical administrationwill be accomplished using a patch either of the reservoir and porousmembrane type or of a solid matrix variety. In either case, the activeagent is delivered continuously from the reservoir or microcapsulesthrough a membrane into the active agent permeable adhesive, which is incontact with the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent is administered to the recipient. In the case ofmicrocapsules, the encapsulating agent may also function as themembrane. The transdermal patch may include the compound in a suitablesolvent system with an adhesive system, such as an acrylic emulsion, anda polyester patch. The oily phase of the emulsions of this invention maybe constituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it may comprise a mixture of at leastone emulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make-up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate,among others. The choice of suitable oils or fats for the formulation isbased on achieving the desired cosmetic properties, since the solubilityof the active compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters may be used.These may be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredients are dissolved or suspended insuitable carrier, especially an aqueous solvent for the activeingredients. The antiinflammatory active ingredients are preferablypresent in such formulations in a concentration of 0.5 to 20%,advantageously 0.5 to 10% and particularly about 1.5% w/w. Fortherapeutic purposes, the active compounds of this combination inventionare ordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered per os, the compoundsmay be admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. Other adjuvants and modes ofadministration are well and widely known in the pharmaceutical art.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient. The daily dose can be administered in one tofour doses per day. In the case of skin conditions, it may be preferableto apply a topical preparation of compounds of this invention to theaffected area two to four times a day.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

General Procedure

Representative synthetic procedures for the preparation of compounds ofthe invention are outlined below in the following schemes. Those havingskill in the art will recognize that the starting materials and reactionconditions may be varied, the sequence of the reactions altered, andadditional steps employed to produce the desired compounds. In somecases, protection of certain reactive functionalities may be necessaryto achieve some of the above transformations. In general, the need forsuch protecting groups as well as the conditions necessary to attach andremove such groups will be apparent to those skilled in the art oforganic synthesis.

Unless otherwise indicated, R₁, R₂, X, X′, G, G₁, G₂, T, T₁, T₂, n1, n2,n3, and n4 carry the definitions set forth above in connection withformulae I-VI. The variable LG represents a suitable leaving groupincluding, but not limited to, halogen, toluenesulfonyl,methanesulfonyl, trifluoromethanesulfonyl, 2,2,2-trifluoroethoxy,N-hydroxysuccinimidyl, and the like.

EXAMPLES

The preparation of the compounds of the invention is illustrated furtherby the following examples, which are not to be construed as limiting theinvention in scope or spirit to the specific procedures and compoundsdescribed in them. Representative methods for synthesizing compounds ofthe invention are presented below. It is understood that the nature ofthe substituents required for the desired target compound oftendetermines the preferred method of synthesis.

Example 1

Synthesis of N,N′-dioleoyl tetrakis(aminomethyl)methane (“dioleoylcrossamine”)

Tetrakis(aminomethyl)methane is prepared by a known procedure (Adil, K.et al., Solid State Sciences, 6 (2004), 1229-1235). A 50 mL flask isequipped with a reflux condenser and a magnetic stirrer. The flask ischarged with tetrakis(aminomethyl)methane tetrachloride (800 mg, 2.88mmol), methanol (10 mL) and NaOMe/MeOH solution (2.10 g of 5.457M, 11.50mmol). The mixture is stirred and refluxed for 4 hours, then cooled.Methanol solution is decanted from the inorganic salts, and the saltsare re-suspended in absolute ethanol (15 mL). The suspension iscentrifuged, and the combined organics are concentrated in vacuo. Theresidue is dissolved in methylene chloride (15 mL) and filtered using asyringe (0.45 micron filter) from the remaining inorganic salts.Concentration of the filtrates affords free tetrakis(aminomethyl)methanein a quantitative yield as a white semi-solid material (the residualalcohol is estimated by NMR). NMR (D₂O) δ 2.9 (s, CH₂)

The obtained tetrakis(aminomethyl)methane (420 mg, 90% purity, 2.86mmol) is dissolved in absolute ethanol (15 mL) and trifluoroacetic acid(400 mg, 3.50 mmol) is added, followed by 2,2,2-trifluoroethyloleate(2.08 g, 5.72 mmol) [vide infra, Example 3]. The homogeneous mixture isallowed to react for 72 hrs at room temperature, and concentrated invacuo. The residue is dissolved in MeOH/water (10%)/trifluoroacetic acid(0.1%) (40 mL), and pH is adjusted to ca. pH 2 with trifluoroaceticacid. The residue is purified by chromatography on C-8 modified silica,using NeOH/water (10%)/tri-fluoroacetic acid (0.1%) as eluent.N,N′-dioleoyl tetrakis(aminomethyl)methane (“dioleoyl crossamine”) isisolated as bistrifluoroacetic salt (0.9 g). MS (TFA salt) 661 [M+1];NMR (CDCl₃) δ 8.5 (br. 6H), 8.05 (br, 2H), 5.37 (m, 4H), 3.07 and 2.95(poorly resolved, 4H each); 2.15 (t, 4H), 2.01 (m, 8H), 1.35 (m, 48H),0.9 (t, 6H)

Minor amounts of 2, 3, and 4 are produced when the reaction is carriedunder higher pH (e.g. without trifluoroacetic acid or in the presence ofNaOMe), and those can be readily removed (e.g. chromatography):

Example 2

2.1. Folate-PEG3400-COOH

170 mg of H₂N-PEG3400-COOH (manufactured by Laysan) is dissolved in dryDMSO (2 mL) and dry chloroform (2 mL). Hunig's base (15 μL) is added,followed by addition of Folic acid-NHS ester (60 mg, prepared by a knownprocedure Lee, R. J.; Low, P. S.; “Methods in Molecular Medicine”, 25,69-76, April 2000). Additional 80 mg of Folic acid-NHS is added as asolution in DMSO (2.5 mL). The stirred mixture is kept at roomtemperature for 24 hrs; the chloroform is removed in vacuo, and theresidue is diluted with deionized water (60 mL). The resultinghomogeneous yellow solution is dialyzed against deionized water, with afew drops of TFA to pH 2-3, using 3500 D cutoff membrane. After sixdialysis bath changes the yellow solution is collected and concentratedin vacuo to afford Folate-PEG3400-COOH.

2.2. Coupling with Folate-PEG3400-COOH to obtain “folate-PEG-dioleoylcrossamine” (5)

150 mg of Folate-PEG3400-COOH is dissolved in dry DMSO (2 mL) and NHS (9mg) is added, followed by DCC (16 mg). The mixture is allowed to reactin the dark for 20 hrs to obtain Folate-PEG3400-COONHS. To thissolution, 30 mg of N,N′-dioleoyl tetrakis(aminomethyl)methane in dryDMSO (2 mL) is added. After the reaction mixture is reacted for 24 hrs,it is diluted with deionized water (60 mL). The resulting homogeneousyellow solution is dialyzed against deionized water using 3500 D cutoffbag, to afford Folate-PEG-ylated N,N′-dioleoyltetrakis(aminomethyl)methane (“folate-PEG-dioleoyl crossamine”) as ca.50% mixture with unreacted Folate-PEG3400-COOH.

Example 3

Synthesis of N,N′-dioleoyl tris(aminoethyl)amine (“dioleoyl monoamine”)(6)

Oleoyl chloride (75 g, 250 mmol) is heated at reflux with2,2,2-trifluoroethanol (60 g, 600 mmol) for 16 hrs. After the evolutionof HCl ceased, excess trifluoroethanol is removed in vacuo. Vacuumdistillation of the residue afforded 87.6 g of 2,2,2-trifluoroethyloleate as colorless liquid, bp 150°/0.3.

Tris-(2-aminoethyl)amine (1.45 g, 10 mmol) is dissolved in 20 mL ofethanol. To this solution 2,2,2-trifluoroethyl oleate (5.6 g) is added,and the reaction mixture is refluxed for 24 hrs. The mixture isconcentrated in vacuo, the residue is dissolved in 90% acetonitrile/10%water and acidified to pH 2-3 with trifluoroacetic acid (TFA).Reverse-phase chromatography of this solution on C8 silica (eluent 89.9%acetonitrile/10% water/0.1% TFA) affords 1.8 g of N,N′-dioleoyltris(aminoethyl)amine (“dioleoyl monoamine”) as its trifluoroaceticsalt. MS (TFA salt) 675 [M+1]; NMR (CDCl₃) δ 7.4 (br, 2H), 5.37 (m, 4H),3.55, 3.37 and 3.05 (poorly resolved, 12H total), 2.15 (t, 4H), 2.01 (m,8H), 1.35 (m, 48H), 0.9 (t, 6H).

Example 3A

Preparation of Methyl-dioleoyl monoamine (6.1)

The title compound is prepared by methylation of the product of Example3, N,N′-dioleoyl tris(aminoethyl)amine {“dioleoyl monoamine”), with anexcess of iodomethane.

Example 4

Synthesis of N,N′-dioleoyl-N″-acetylsalicyloyl tris(aminoethyl)amine (7)

N,N′-dioleoyl-tris(aminoethyl)amine trifluoroacetate (80 mg, 100 μmol)is dissolved in 5 mL chloroform and treated with 3 mL of 10% aq. K₂CO₃solution. The organic phase is separated, dried and concentrated invacuo to afford N,N′-dioleoyl-tris(aminoethyl)amine as the free base.This material is dissolved in 3 mL of dry chloroform and the solution istreated with acetylsalycyloylchloride (20 mg, 100 μmol) to afford thetitle compound.

Example 5

Coupling with Folate-PEG3400-COOH to obtain “folate-PEG-dioleoylmonoamine” (8)

Folate-dioleoyl monoamine is obtained essentially as described inExample 2 from Folate-PEG3400-COOH and dioleyl monoamine.

Example 6

Complex of dioleoyl crossamine with siRNA; formation of nanoparticles

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isdioleoyl crossamine. siRNA (3 mg/mL) and dioleoyl crossamine (5 mg/mL)solutions are separately prepared in water for injection andsubsequently diluted in 5% dextrose to 0.3 mg/mL for siRNA and 1.5 mg/mLfor dioleoyl crossamine. The siRNA in dextrose solution is added todioleoyl crossamine in dextrose solution using a micropipette to acharge ratio of 5:1 (positive:negative). The formulation is incubatedfor 15 minutes at room temperature to allow the complexes to form.

Example 7

Complex of dioleoyl crossamine with DNA; formation of nanoparticles

The nucleic acid used is a plasmid DNA encoding for luciferase gene. Thecationic lipid used is dioleoyl crossamine. The plasmid DNA (3 mg/mL)and dioleoyl crossamine (5 mg/mL) solutions are separately prepared inwater for injection, and subsequently diluted in 5% dextrose to 0.3mg/mL for plasmid DNA and 1.5 mg/mL for dioleoyl crossamine. The plasmidDNA in dextrose solution is added to dioleoyl crossamine in dextrosesolution using a micropipette to a charge ratio of 5:1(positive:negative). The formulation is incubated for 15 minutes at roomtemperature to allow the complexes to form.

Example 8

Complex of Folate-PEG-dioleoyl crossamine with siRNA

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isdioleoyl crossamine conjugated to folate ligands or co-formulated withfolated dioleoyl crossamine at different molar ratios (1:1), (2:1),(4:1), or (10:1). The co-formulant is added to dioleoyl crossaminechloroform solution, and liposome preparation is performed as describedin Example 9. The siRNA (3 mg/mL) and folate-PEG-dioleoyl crossamine (5mg/mL) solutions are separately prepared in water for injection andsubsequently diluted in 5% dextrose to 0.3 mg/mL for siRNA and 1.9 mg/mLfor folate-PEG-dioleoyl crossamine. The siRNA in dextrose solution isadded to folate-PEG-dioleoyl crossamine in dextrose solution using amicropipette to a charge ratio of 5:1 (positive:negative). Theformulation is incubated for 15 minutes at room temperature to allow thecomplexes to form.

Example 9

Complex of dioleoyl monoamine and siRNA; formation of nanoparticles

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isdioleoyl monoamine. siRNA (3 mg/mL) and dioleoyl monoamine (5 mg/mL)solutions are separately prepared in water for injection, andsubsequently diluted in 5% dextrose to 0.3 mg/mL for siRNA and 1.5 mg/mLfor dioleoyl monoamine. The siRNA in dextrose solution is added to thedioleoyl monoamine in dextrose solution using a micropipette to a chargeratio of 5:1 (positive negative). The formulation is incubated for 15minutes at room temperature to allow the complexes to form.

Example 10

Complex of dioleoyl monoamine and DNA; formation of nanoparticles

The nucleic acid used is a plasmid DNA encoding for luciferase gene andthe cationic lipid used is dioleoyl monoamine. The plasmid DNA (3 mg/mL)and dioleoyl monoamine (5 mg/mL) solutions are separately prepared inwater for injection, and subsequently diluted in 5% dextrose to 0.3mg/mL for the plasmid DNA and 1.5 mg/mL for dioleoyl monoamine. Theplasmid DNA in dextrose solution is added to the dioleoyl monoamine indextrose solution using a micropipette to a charge ratio of 5:1(positive:negative). The formulation is incubated for 15 minutes at roomtemperature to allow the complexes to form.

Example 11

Preparation of encapsulated siRNA with dioleoyl crossamine

This example illustrates liposomal encapsulation of siRNA using dioleoylcrossamine. The siRNA used is a double stranded sequence of nucleotidesintended to produce a knock-down of endogenous vascular endothelialgrowth factor (VEGF) transcripts and protein levels. The cationic lipidused is dioleoyl crossamine. To prepare the capsulation liposomes, thelipids are dissolved and mixed in chloroform to assure a homogeneousmixture of lipids. The organic solvent is removed by rotary evaporationyielding a thin lipid film on the sides of a round flask. Chloroform isfurther evaporated by placing the round flask on a vacuum pumpovernight. The resulting lipid film is dissolved initially in 100%ethanol then brought to 50%. siRNA dissolved in water is added toliposomes/ethanol solution. Ethanol is evaporated from theliposomes/siRNA mixture by a rotary evaporation system. The resultingnanoparticles are suspended in 5% dextrose by adding and equal amount of10% dextrose to the encapsulated siRNA particles.

Example 12

Preparation of encapsulated siRNA with folate-PEG-dioleoyl crossamine

This example illustrates liposomal encapsulation of siRNA usingfolate-PEG-dioleoyl crossamine. The siRNA used is a double strandedsequence of nucleotides intended to produce a knock-down of endogenousvascular endothelial growth factor (VEGF) transcripts and proteinlevels. The cationic lipid used is folate-PEG-dioleoyl crossamine. Toprepare the capsulation liposomes, the lipids are dissolved and mixed inchloroform to assure a homogeneous mixture of lipids. The organicsolvent is removed by rotary evaporation yielding a thin lipid film onthe sides of a round flask. Chloroform is further evaporated by placingthe round flask on a vacuum pump overnight. The resulting lipid film isdissolved initially in 100% ethanol then brought to 50%. siRNA dissolvedin water is added to liposomes/ethanol solution. Ethanol is evaporatedfrom the liposomes/siRNA mixture by a rotary evaporation system. Theresulting nanoparticles are suspended in 5% dextrose by adding and equalamount of 10% dextrose to the encapsulated siRNA particles.

Example 13

Preparation of siRNA transfection complexes with dioleoyl monoamine withco-formulants

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The dioleoyl monoamine isformulated with other lipids at a molar ratio of (4:1). The lipids usedare 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550], 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-Lactosyl,and Cholesterol. The co-formulants are added to dioleoyl monoamine inchloroform. Liposomes are prepared as previously described in Example 9.The siRNA (3 mg/mL) and dioleoyl monoamine (5 mg/mL) solutions areseparately prepared in water for injection, and subsequently diluted in5% dextrose to 0.3 mg/mL for siRNA and 1.9 mg/mL for dioleoyl monoamine.The siRNA in dextrose solution is added to the dioleoyl monoamine indextrose solution using a micropipette to a charge ratio of 5:1(positive negative). The formulation is incubated for 15 minutes at roomtemperature to allow the complexes to form.

Example 14

Preparation of DNA transfection complexes with dioleoyl monoamine

The nucleic acid used is a plasmid DNA encoding for luciferase gene andthe dioleoyl monoamine lipid. The plasmid DNA (3 mg/mL) and dioleoylmonoamine (5 mg/mL) solutions are separately prepared in water forinjection and subsequently diluted in 5% dextrose to 0.3 mg/mL forplasmid DNA and 1.9 mg/mL for dioleoyl monoamine. The plasmid DNA indextrose solution is added to the dioleoyl monoamine in dextrosesolution using a micropipette to a charge ratio of 5:1(positive:negative). The formulation is incubated for 15 minutes at roomtemperature to allow the complexes to form.

Example 15

Preparation of DNA transfection complexes with dioleoyl monoamine withco-formulants

The nucleic acid used is a plasmid DNA encoding for luciferase gene andthe cationic lipid used is dioleoyl monoamine. The cationic lipid usedin this example is dioleoyl monoamine formulated with other lipids at amolar ratio of (4:1). The lipids used are1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550], 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-Lactosyl,and Cholesterol. The co-formulants are added to dioleoyl monoaminechloroform solution. Liposomes are prepared as previously described inExample 9. The DNA (3 mg/mL) and dioleoyl monoamine co-formulants (5mg/mL) solutions are separately prepared in water for injection, andsubsequently diluted in 5% dextrose to 0.3 mg/mL for DNA and 1.9 mg/mLfor dioleoyl monoamine. The DNA in dextrose solution is added to thedioleoyl monoamine in dextrose solution using a micropipette to a chargeratio of 5:1 (positive:negative). The formulation is incubated for 15minutes at room temperature to allow the complexes to form.

Example 16

Preparation of siRNA transfection complexes with dioleoyl monoamineco-formulated with folate-PEG-dioleoyl monoamine

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isdioleoyl monoamine co-formulated with folate-PEG-dioleoyl monoamine atdifferent molar ratios (1:1), (2:1), (4:1), or (10:1). The co-formulantis added to dioleoyl monoamine chloroform solution. Liposomes areprepared as previously described in Example 9. The siRNA (3 mg/mL) anddioleoyl monoamine/folate-PEG-dioleoyl monoamine (5 mg/mL) solutions areseparately prepared in water for injection, and subsequently diluted in5% dextrose to 0.3 mg/mL for siRNA and 1.9 mg/mL for dioleoyl monoamine.The siRNA in dextrose solution is added to the dioleoyl monoamine indextrose solution using a micropipette to a charge ratio of 5:1(positive:negative). The formulation is incubated for 15 minutes at roomtemperature to allow the complexes to form.

Example 17

Preparation of encapsulated siRNA with dioleoyl monoamine liposomes

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isdioleoyl monoamine. To prepare the capsulation liposomes, the lipids aredissolved and mixed in chloroform to assure a homogeneous mixture oflipids. The organic solvent is removed by rotary evaporation yielding athin lipid film on the sides of a round flask. Chloroform is furtherevaporated by placing the round flask on a vacuum pump overnight. Theresulting lipid film is dissolved initially in 100% ethanol then broughtto 50%. siRNA dissolved in water is added to liposomes/ethanol solution.Ethanol is evaporated from the liposomes/siRNA mixture by a rotaryevaporation system. The resulting nanoparticles are suspended in 5%dextrose by adding and equal amount of 10% dextrose to the encapsulatedsiRNA particles.

Example 18

Preparation of encapsulated siRNA with folate-PEG-dioleoyl monoamineliposomes

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipid used isfolate-PEG-dioleoyl monoamine. To prepare the capsulation liposomes, thelipids are dissolved and mixed in chloroform to assure a homogeneousmixture of lipids. The organic solvent is removed by rotary evaporationyielding a thin lipid film on the sides of a round flask. Chloroform isfurther evaporated by placing the round flask on a vacuum pumpovernight. The resulting lipid film is dissolved initially in 100%ethanol then brought to 50%. siRNA dissolved in water is added toliposomes/ethanol solution. Ethanol is evaporated from theliposomes/siRNA mix by a rotary evaporation system. The resultingnanoparticles are suspended in 5% dextrose by adding and equal amount of10% dextrose to the encapsulated siRNA particles.

Example 19

Transfection activity of siRNA and dioleoyl crossamine complexes

The transfection activity of siRNA and dioleoyl crossamine complexes isdetermined in vitro as follows. Transfection complexes containing mVEGFor luciferase siRNA constructs are prepared by methods described inExample 9. SCCVII cells (0.5×10⁵ cells/well; murine squamous cellcarcinomas) are seeded into 24-well tissue culture plates in 10% fetalbovine serum (FBS). Each well is incubated for 6 hours with 0.5 μg ofcomplexed siRNA in absence or presence of FBS in a total volume of 250μL of Dulbecco Modified Eagle's Minimal Essential Medium (DMEM). Whenthe incubation period is concluded for the cells lacking FBS in theirmedium, 250 μL of DMEM supplemented with 20% FBS is added to thetransfected cells and incubated further for another 40 hours. To cellswith FBS in their transfection medium, 250 μL of DMEM supplemented with10% FBS is added to the transfected cells and incubated further foranother 40 hours. At the end of the incubation period, knock down ofmVEGF protein and transcripts are evaluated in the cell culture medium(protein) or cell lysate (transcripts). For measurement of mVEGF proteinlevels, cell culture medium is directly analyzed by mVEGF ELISA assay.For mVEGF transcripts analysis, cells are lysed using Tri-reagent andthen quantified for the level of mVEGF transcripts using qRT-PCRdetection kit.

Example 20

Transfection activity of encapsulated siRNA with dioleoyl crossamine

This example illustrates liposomal encapsulation of siRNA using dioleoylmonoamine, dioleoyl crossamine, or a mixture of both cationic agents.The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. The cationic lipids used aredioleoyl monoamine (Example 3), dioleoyl crossamine (Example 1), or amixture of both. To prepare the capsulation liposomes, the lipids aredissolved and mixed in chloroform to assure a homogeneous mixture oflipids. The organic solvent is removed by rotary evaporation yielding athin lipid film on the sides of a round flask. Chloroform is furtherevaporated by placing the round flask on a vacuum pump overnight. Theresulting lipid film is dissolved initially in 100% ethanol then broughtto 50%. siRNA dissolved in water is added to liposomes in ethanolsolution. Ethanol is evaporated from the liposomes/siRNA mixture by arotary evaporation system. The resulting nanoparticles are suspended in5% dextrose by adding and equal amount of 10% dextrose to theencapsulated siRNA particles. SCCVII cells (0.5×10⁵ cells/well) areseeded into 24-well tissue culture plates in 10% FBS. Each well isincubated for 6 hours with 0.5 μg of complexed siRNA in absence orpresence of FBS in a total volume of 250 μL of DMEM. When the incubationperiod is concluded for the cells lacking FBS in their medium, 250 μL ofDMEM supplemented with 20% FBS is added to the transfected cells andincubated further for another 40 hours. To cells with FBS in theirtransfection medium, 250 μL of DMEM supplemented with 10% FBS is addedto the transfected cells and incubated further for another 40 hours. Atthe end of the incubation period, knock down of mVEGF protein andtranscripts are evaluated is measured in the cell culture medium(protein) or cell lysate (transcripts). For measurement of mVEGF proteinlevels, cell culture medium is directly analyzed by mVEGF ELISA assay.For mVEGF transcripts analysis, cells are lysed using Tri-reagent andthen quantified for the level of mVEGF transcripts using qRT-PCRdetection kit.

Example 21

Transfection activity of encapsulated siRNA with folate-PEG-dioleoylcrossamine

This example illustrates liposomal encapsulation of siRNA usingfolate-PEG-dioleoyl monoamine, folate-PEG-dioleoyl crossamine, or amixture of both cationic agents. The siRNA used is a double strandedsequence of nucleotides intended to produce a knock-down of endogenousvascular endothelial growth factor (VEGF) transcripts and proteinlevels. The cationic lipids used are folate-PEG-dioleoyl monoamine(Example 5), folate-PEG-dioleoyl crossamine liposomes (Example 2), ormixture of both. To prepare the capsulation liposomes, the lipids aredissolved and mixed in chloroform to assure a homogeneous mixture oflipids. The organic solvent is removed by rotary evaporation yielding athin lipid film on the sides of a round flask. Chloroform is furtherevaporated by placing the round flask on a vacuum pump overnight. Theresulting lipid film is dissolved initially in 100% ethanol then broughtto 50%. siRNA dissolved in water is added to liposomes in ethanolsolution. Ethanol is evaporated from the liposomes/siRNA mixture by arotary evaporation system. The resulting nanoparticles are suspended in5% dextrose by adding and equal amount of 10% dextrose to theencapsulated siRNA particles. SCCVII cells (0.5×10⁵ cells/well) areseeded into 24-well tissue culture plates in 10% FBS. Each well isincubated for 6 hours with 0.5 μg of complexed siRNA in absence orpresence of FBS in a total volume of 250 μL of DMEM. When the incubationperiod is concluded for the cells lacking FBS in their medium, 250 μL ofDMEM supplemented with 20% FBS is added to the transfected cells andincubated further for another 40 hours. To cells with FBS in theirtransfection medium, 250 μL of DMEM supplemented with 10% FBS is addedto the transfected cells and incubated further for another 40 hours. Atthe end of the incubation period, knock down of mVEGF protein andtranscripts are evaluated is measured in the cell culture medium(protein) or cell lysate (transcripts). For measurement of mVEGF proteinlevels, cell culture medium is directly analyzed by mVEGF ELISA assay.For mVEGF transcripts analysis, cells are firstly, lysed usingTri-reagent and then quantified for the level of mVEGF transcripts usingqRT-PCR detection

Example 22

Uterine VEGF protein knockdown following intravaginal administration ofdioleoyl monoamine complexed siRNA

Two days preceding siRNA administration female ICR mice (17-22 grams)are given two daily sub-cutaneous administrations of progesterone (0.5mg dissolved in sesame oil) to normalize estrus cycling in the animals.Mice are then intravaginally administered formulated siRNA targetingVEGF transcript or a non-targeting siRNA control. siRNA is complexedwith dioleoyl monoamine at a 5:1 N:P charge ratio. A total of 9 μg siRNAis administered in a final volume of 30 μl (0.3 mg/mL). At 48 hoursafter administration, the animals are euthanized and the vagina/cervixand uterus (including uterine horns) are collected dissected free ofother tissue and stored frozen in liquid N₂. Only tissues from animalsthat had a gross appearance consistent with progesterone treatment areused for analysis. Tissue is homogenized in lysis buffer and VEGFproteins determinations made by ELISA.

Example 23

Protein and transcript knockdown of growth factor in ascites andintraperitonel tumor nodules in animals with disseminated peritonealmalignancies following administration of crossamine siRNA

Formulations containing siRNA and dioleoyl crossamine are administeredto animals with disseminated ovarian cancer in an effort to decreaseVEGF levels that have been associated with disease progression. Toinduce disseminated ovarian cancer, female C57BL/6 mice are implanted IPwith 2.5×10⁶ ID8 (murine ovarian carcinoma) cells. At a predeterminedday after tumor implant mice are injected with 200 μg siVEGF (ornon-coding control siRNA) formulated with dioleoyl crossamine at an N:Pratio of 5:1. Starting at 24 hours after treatment animals areeuthanized, tumor and ascites fluid are harvested from the peritonealcavity and analyzed for VEGF protein and transcript levels. For ascitestranscript analysis, samples are initially subjected to a red blood celllysis protocol and enriched for nucleated cells prior to RNA isolation.

Example 24

Tumor targeted siRNA using folate-PEG-dioleoyl crossamine

VEGF siRNA formulated with folate-PEG-dioleoyl crossamine is deliveredintravenously or intraperitoneally into mice bearing subcutaneous SCCVIItumors. At specified time points after administration tumors areharvested and transcript and protein determination of the targetedtranscript is determined.

Example 25

Enhanced systemic uptake of siRNA using peptide modified dioleoylcrossamine

siRNA is formulated with dioleoyl crossamine that has been functionallymodified to target integrin receptor by the addition of a peptide havinga conserved Arg-Gly-Asp motif. The complex is delivered intravenously orintraperitoneally to tumor bearing animals. At specified time pointsafter administration various tissues are harvested and transcript andprotein determination of the targeted transcript/protein is determined.

Example 26

Protein knock-down following in vitro transfection of complexed siRNAwith dioleoyl monoamine delivery reagent.

The transfection activity of siRNA and dioleoyl monoamine deliveryreagent conjugate complexes is determined in vitro. Transfectioncomplexes containing simVEGF siRNA construct are prepared by methodspreviously described in Example 9. SCCVII cells (0.5×10⁵ cells/well) areseeded into 24-well tissue culture plates in 10% FBS. Each well isincubated for 6 hours with 0.5 μg of complexed siRNA in a total volumeof 250 μL of DMEM. When the incubation period is concluded, 250 μL ofDMEM supplemented with 20% FBS is added to the transfected cells andincubated further for another 40 hours. At the end of the incubationperiod, supernatant mVEGF protein determinations are made. The resultsindicate a significant (˜95%) knockdown of VEGF protein (FIG. 1).

Example 27

Protein knock-down following in vitro transfection of complexed siRNAwith dioleoyl monoamine and methyl-dioleoyl monoamine delivery reagents.

The transfection activity of siRNA complexes with dioleoyl monoamine(Example 3) or methyl-dioleoyl monoamine (Example 3A) delivery reagentsis determined in vitro. Transfection complexes containing simVEGF siRNAconstruct are prepared by methods previously described. SCCVII cells(0.5×10⁵ cells/well) are seeded into 24-well tissue culture plates in10% FBS. Each well is incubated for 6 hours with 0.5 μg of complexedsiRNA in a total volume of 250 μL of DMEM. When the incubation period isconcluded, 250 μL of DMEM supplemented with 20% FBS is added to thetransfected cells and incubated further for another 40 hours. At the endof the incubation period, supernatant mVEGF protein determinations aremade as described above. The results indicate a significant (˜95%)knockdown of VEGF protein (FIG. 2).

Example 28

Protein and transcripts knock-down following in vitro transfection ofcomplexed siRNA with folate-PEG-dioleoyl monoamine

The transfection activity of siRNA and folate-PEG-dioleoyl monoamineconjugate complexes is determined in vitro. Transfection complexescontaining simVEGF siRNA construct are prepared by methods previouslydescribed. SCCVII cells (0.5×10⁵ cells/well) are seeded into 24-welltissue culture plates in 10% FBS. Each well is incubated for 6 hourswith 0.5 μg of complexed siRNA in absence or presence of folatesubstrate at variable concentrations in a total volume of 250 μL ofDMEM. When the incubation period is concluded, 250 μL of DMEMsupplemented with 20% FBS is added to the transfected cells andincubated further for another 40 hours. At the end of the incubationperiod, mVEGF protein and mVEGF transcripts levels are measured from thecell culture medium and cell lysates respectively. For measurement ofmVEGF protein levels, cell culture medium is directly analyzed by mVEGFELISA assay. For mVEGF transcripts analysis, cells are lysed usingTri-reagent and then quantified for the level of mVEGF transcripts usingqRT-PCR detection kit. The percentage of VEGF protein and transcriptknockdown that occurs relative to cells that have been transfected withnon silencing control siRNA is calculated.

Example 29

Caveolin-1 transcript knockdown in lung and liver followingadministration of siRNA complexed with dioleoyl monoamine deliveryreagent.

Female ICR mice (17-22 grams) are injected intravenously with formulatedsiRNA targeting Caveolin-1 (Cav-1) transcript or a non-targeting siRNAcontrol. siRNA is complexed with dioleoyl monoamine delivery reagent ata 10:1 ratio. A total of 100 μg siRNA is injected in a final volume of200 μL (0.5 mg/mL). At 48 hours after injection the animals areeuthanized and lungs and livers are harvested for Cav-1 transcriptanalysis using qRTPCR. Transcript levels are normalized to β-actin as aninternal control. Results (FIG. 3) indicate a significant decrease intarget specific Cav-1 transcript levels in both lungs (76% knockdown)and livers (62% knockdown). For each group, n=5.

Example 30

VEGF transcript knockdown resulting in tumor inhibition followingdioleoyl monoamine formulated siVEGF injected into subcutaneous tumorsin mice.

In this example tumors were implanted into the hind flanks of CH₃ miceby injecting 5×10⁵ squamous cell carcinoma cells. Tumors were allowed togrow until they reached a size of ˜40 mm³. Tumors were then injectedwith a commercially available siRNA targeting VEGF that was formulatedwith dioleoyl monoamine at a 5:1 N:P ratio or with a similarlyformulated non-silencing control siRNA. The final concentration of siRNAwas 0.3 mg/mL. A total of 30 μl of the formulated siRNA solution wasinjected into tumors and injections were repeated every 3-4 days for atotal of 6 injections. The tumors from some animals were harvested 48hours after the second formulated siRNA injection for transcriptanalysis. Results from this study (FIG. 4) indicate that administrationof the VEGF siRNA resulted in a 32% decrease in VEGF transcript relativeto the non-silencing control group. One week following the last tumorinjection there was a 31% decrease in tumor volume in the VEGF siRNAgroup relative to non-silencing siRNA and a 57% decrease relative tountreated control animals. The VEGF siRNA treatments further resulted ina 13% improvement in median survival of animals relative to either thenon-silencing control (siNON) or untreated groups.

Example 31

Preparation of concentrated siRNA transfection complexes with dioleoylmonoamine by filtration

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. Liposomes of the cationic lipidsare prepared as previously described in Example 9. The siRNA (20 mg/mL)and dioleoyl monoamine (6.4 mg/mL) solutions are separately prepared inwater for injection. 25 μL of the siRNA is combined with 500 μL ofdioleoyl monoamine solution. Subsequently, the complex is diluted in 5%dextrose to 0.03 mg/mL of siRNA. Diluted complex is transferred into25-50 kDa centrifugal filter unit and centrifuged for several minutes toreach the desired siRNA concentration in the retentate.

Example 32

Preparation of concentrated siRNA transfection complexes with dioleoylcrossamine by filtration

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knock-down of endogenous vascular endothelial growth factor(VEGF) transcripts and protein levels. Liposomes of the cationic lipidsare prepared as previously described in Example 9. The siRNA (20 mg/mL)and dioleoyl crossamine (5 mg/mL) solutions are separately prepared inwater for injection. 25 μL of the siRNA is combined with 500 μL ofdioleoyl monoamine solution. Subsequently, the complex is diluted in 5%dextrose to 0.03 mg/mL of siRNA. Diluted complex is transferred to 25-50kDa centrifugal filter unit and centrifuged for several minutes untilthe desired siRNA concentration in the retentate is reached.

Example 33

Determination of particle size and zeta potential of nanoparticles

Dioleoyl crossamine/siRNA and dioleoyl monoamine/siRNA complexes areformulated as in Examples 6 and 9, and are diluted in 50 mM NaCl toappropriate concentrations. Samples are then added to a polystyrenecuvet and measured for particle size and zeta potential using a90Plus/BI-MAS particles sizer. Observed particle sizes are between80-400 nm and observed zeta potentials are between +10 to +40 mV.

Example 34

Synthesis of adi-[2-(oleoylamino)ethyl][2-[(methoxydodecaethyleneglycolcarbonylamino)ethyl]amine“mPEG-dioleoyl monoamine” (9)

Methoxy(dodecaethylene glycol) (MW 550, polydisperse, containingapproximately 12 ethylene glycol units, 720 mg, 1.30 mmol) is dissolvedin 8 mL of dry toluene. To this phosgene solution in toluene (4 mL of a2 M solution, 8 mmol) is added. The reaction mixture is stirred at roomtemperature for 3 hours, and then concentrated in vacuuo at roomtemperature, affording 810 mg (1.31 mmol) of crudemethoxy(dodecaethyleneglycol)chloroformate.

The above methoxy(dodecaethyleneglycol)chloroformate (810 mg, 1.31 mmol)is dissolved in 6.313 g of dry methylene chloride. dioleoyl monoamine isconverted to the free base by treatment with potassium carbonate andextraction with methylene chloride. The organic phase is dried to give adry oily material (700 mg, 1.04 mmol), which is re-dissolved in 6 mL drymethylene chloride. 5.66 g of the Methoxy(dodecaethyleneglycol)chloroformate solution (1.04 mmol of mPEG chloroformate) is addedwith stirring to the dioleoyl monoamine free base solution. The mixtureis stirred for 3 hours at room temperature, and then concentrated andpurified chromatographically using silica gel (3 to 15% methanol inmethylene chloride gradient elution). The purification affordsPEG-dioleoyl monoamine (830 mg, 0.664 mmol).

Example 35

Preparation of a fluorescent taggant-carrying monoamine

The sulforhodamine B acyl chloride [Fluka, 115 mg, 0.2 mmol] isdissolved in 7 mL of dry chloroform. This solution is added to the wellstirred solution of dioleoyl monoamine free base [140 mg, ca. 0.2 mmol,prepared as above by a treatment of the chloroform solution of its TFAsalt with potassium carbonate] in 5 mL of dry chloroform. The mixture isstirred for 3 hours at room temperature, and then concentrated andpurified chromatographically using 2000 micron thick layer silica gelpreparative plate [subsequent elutions in 3 to 15% methanol in methylenechloride]. The purification affords [rhodamine B sulphonyl]dioleoylmonoamine (120 mg, 0.1 mmol).

Example 36

Synthesis of α-lactobionylamido-ω-propionicacidundecaethyleneglycol-di-[2-(oleoylamino)ethyl](2-aminoethyl)amine“lactobionyl-dioleoyl monoamine” (10)

Fmocamino-ω-propionic acid-undecaethyleneglycol linker (Fmoc-PEG₁₁-COOH)(100 mg, 0.119 mmol) and p-nitrophenol (18 mg, 0.129 mmol) is dissolvedin 1 mL methylene chloride. DCC (27 mg, 0.131 mmol) is dissolved in 1 mLmethylene chloride and added to the stirring PEG/p-nitrophenol solution.The reaction is allowed to proceed at room temperature for 3 hours,after which DCU is removed using a 0.45 μm syringe filter. Dioleoylmonoamine is converted to the free base by treatment with potassiumcarbonate and extraction with methylene chloride. The organic phase isdried to give 75 mg (0.111 mmol) dry material which is redissolved in0.5 mL methylene chloride. This solution is added to thep-nitrophenol-activated α-Fmocamino-ω-propionicacid-undecaethyleneglycol acid solution. The reaction is allowed toproceed for 18 hours at room temperature. The crude material is driedand re-dissolved in 1.8 mL dimethylformamide to which 200 μL piperidineis added. The Fmoc cleavage is allowed to proceed for 15 min, afterwhich the dimethylformamide and piperidine are removed under highvacuum. α-Amino-ω-propionicacid-undecaethyleneglycol-di-[2-(oleoylamino)ethyl](2-aminoethyl)amineis isolated by separation on a silica gel column containing 80%methylene chloride in methanol, followed by 75%. The appropriatefractions are dried, giving 75 mg (0.59 mmol) of a brown oily material.

Lactobionic acid is first converted to the corresponding lactone bydehydration at 50° C. in methanol containing a drop of trifluoroaceticacid. α-Amino-ω-propionicacid-undecaethyleneglycol-di-[2-(oleoylamino)ethyl](2-aminoethyl)amine(50 mg, 0.039 mmol) is dissolved in 1.5 mL of methanol containing 15 μLdiisopropylethylamine. To this stirring solution dry lactobionolactone(15 mg, 0.044 mmol) is added. The flask is sealed and the reaction isstirred at 60° C. for 20 hours. A small amount of precipitate is removedby syringe filtration and the α-lactobionylamido-ω-propionicacid-undecaethyleneglycol-di-[2-(oleoylamino)ethyl](2-aminoethyl)amineis dried under high vacuum to give 57 mg (0.035 mmol) of pure material.

Example 37

Synthesis of di-[2-(oleoylamino)ethyl][2-[ω-propionyl-LHRH-octaethyleneglycolpropionylamino)ethyl]amine“LHRH-dioleoyl monoamine” (11)

Octaethyleneglycoldipropionic acid (347 mg, 0.675 mmol) is dissolved in8 mL of dry methylene chloride. To this p-nitrophenol (210 mg, 1.5 mmol)is added, followed by dicyclohexylcarbodiimide (DCC) (313 mg, 1.52mmol). The next day, the reaction mixture is filtered from precipitateddicyclohexylurea, the filtrate concentrated and the title compoundpurified by chromatography on silica (first, elution with ether toremove the unreacted p-nitrophenol, and then with 4% methanol/methylenechloride).

The above octaethyleneglycoldipropionic acid di-p-nitrophenyl ester (510mg, 0.68 mmol) is dissolved in 6 mL of dry methylene chloride.Di-[2-(oleoylamino)ethyl](2-aminoethyl)amine is converted to the freebase by treatment with potassium carbonate and extraction with methylenechloride. The organic phase is dried to give a dry oily material (390mg, 0.58 mmol), which is re-dissolved in 3 mL methylene chloride. Themixture is stirred overnight, and then purified chromatographically,using the same procedure as for the aforementioned di-p-nitrophenylester. After the unreacted di-[2-(oleoylamino)ethyl](2-aminoethyl)aminefraction, di-[2-(oleoylamino)ethyl][2-[(ω-propionic acidoctaethyleneglycolpropionylamino)ethyl]amine p-nitrophenolate (268 mg,0.207 mmol) is collected.

Commercial LHRH peptide (sequence: Ac-QHWSYKLRP-Am, 98 mg of TFA salt,0.078 mmol) is dissolved in 2 mL of dry dimethylformamide and thesolution is evaporated in high vacuum to dry the peptide. The residue isdissolved in 1 mL of dry dimethylformamide; the solution of the abovedi-[2-(oleoylamino)ethyl][2-[ω-propionic acidoctaethyleneglycolpropionylamino)ethyl]amine p-nitrophenolate (135 mg,0.104 mmol) in 1 mL dimethylformamide is added, followed by the additionof triethylamine (0.028 mL, 0.201 mmol). The stirred reaction mixture iskept at room temperature for 17 hours and then concentrated in vacuo.The purification of the target material is accomplished by reverse phasepreparative chromatography using a C8 column and acetonitrile/watergradient elution [30% acetonitrile/water (0.1% TFA) to 90% over 15 min]affords 97 mg of di-[2-(oleoylamino)ethyl][2-[ω-propionyl-LHRHoctaethyleneglycolpropionylamino)ethyl]amine.

Example 38

Synthesis ofdi-[2-(oleoylamino)ethyl][2-[ω-propionyl-RGD-octaethyleneglycolpropionylamino)ethyl]amine “RGD-dioleoyl monoamine” (12)

Octaethyleneglycoldipropionic acid (347 mg, 0.675 mmol) is dissolved in8 mL of dry methylene chloride. To this p-nitrophenol (210 mg, 1.5 mmol)is added, followed by dicyclohexylcarbodiimide (DCC) (313 mg, 1.52mmol). The next day the reaction mixture is filtered from precipitateddicyclohexylurea, the filtrate concentrated and the title compoundpurified by chromatography on silica (first, elution with ether toremove the unreacted p-nitrophenol, and then with 4% methanol/methylenechloride).

The above octaethyleneglycoldipropionic acid di-p-nitrophenyl ester (510mg, 0.68 mmol) is dissolved in 6 mL of dry methylene chloride. Dioleoylmonoamine is converted to the free base by treatment with potassiumcarbonate and extraction with methylene chloride. The organic phase isdried to give a dry oily material (390 mg, 0.58 mmol) which isre-dissolved in mL methylene chloride. The mixture is stirred overnight,and then purified chromatographically using the same procedure as forthe aforementioned di-p-nitrophenyl ester. After the unreacted dioleoylmonoamine fraction, (oleoylamino)ethyl][2-[ω-propionic acidoctaethyleneglycol-propionylamino)ethyl]amine p-nitrophenolate (268 mg,0.207 mmol) is collected.

Commercial RGD peptide [sequence: A*CRGDMFG*CA (2-9 disulfide bridge),117 mg of TFA salt, 0.100 mmol] is dissolved in 3 mL of drydimethylformamide and the solution evaporated in high vacuum to dry thepeptide. The residue is dissolved in 3 mL of dry methanol; the solutionof the above di-[2-(oleoylamino)ethyl][2-[ω-propionic acidoctaethyleneglycol-propionylamino)ethyl]amine p-nitrophenolate (135 mg,0.104 mmol) in 3 mL methylene chloride is added, followed by theaddition of Hunig's base (0.065 mL, 0.370 mmol). The reaction mixture isstirred at room temperature for 72 hours and then concentrated invacuuo. The purification of the target material is accomplished byreverse phase preparative chromatography using a C8 column andacetonitrile/water gradient elution affording 43 mg (0.020 mmol) ofdi-[2-(oleoylamino)ethyl][2-[ω-propionyl-RGD-octaethyleneglycolpropionylamino) ethyl]amine.

Example 39

Preparation of complexed siRNA with dioleoyl monoamine and mPEG-dioleoylmonoamine

The siRNA used is a double stranded sequence of nucleotides intended toproduce a knockdown of endogenous vascular endothelial growth factor(VEGF) transcript and protein levels. The cationic lipids used aremixtures of dioleoyl monoamine (Example 3) and mPEG-dioleoyl monoamine(Example 34). To prepare the liposomes, the lipids are dissolved andmixed in chloroform to assure a homogenous mixture. The organic solventis removed by rotary evaporation yielding a thin lipid film on the wallsof a round-bottom flask. Chloroform is further evaporated by placing theround-bottom flask on a vacuum pump overnight. The resulting lipid filmis rehydrated with distilled water at a desired concentration andvortexed vigorously for several minutes. The solution is placed in abath sonicator for 1 hour and is then filtered through a 200 nm sterilesyringe filter to give the final liposomal solution. siRNA dissolved inwater is added to the liposome solution.

Example 40

Preparation of encapsulated siRNA with dioleoyl monoamine andmPEG-dioleoyl monoamine

This example illustrates liposomal encapsulation of siRNA usingmPEG-dioleoyl monoamine. The siRNA used is a double stranded sequence ofnucleotides intended to produce a knockdown of endogenous targettranscript and protein levels. The cationic lipids used are mixtures ofdioleoyl monoamine (Example 3) and mPEG-dioleoyl monoamine (Example 34).To prepare the encapsulation liposomes, the lipids are dissolved andmixed in chloroform to assure a homogenous mixture. The organic solventis removed by rotary evaporation yielding a thin lipid film on the wallsof a round-bottom flask. Chloroform is further evaporated by placing theround-bottom flask on a vacuum pump overnight. The resulting lipid filmis dissolved initially in 100% ethanol then brought to 50%. siRNAdissolved in water is added to the liposomes in ethanol solution.Ethanol is evaporated from the liposomes/siRNA mixture by a rotaryevaporation system. The resulting nanoparticles are suspended in 5%dextrose by adding an equal amount of 10% dextrose to the encapsulatedsiRNA particles.

Example 41

Transfection activity siRNA and dioleoyl monoamine/mPEG-dioleoylmonoamine complexes

The transfection activity of siRNA and dioleoyl monoamine/mPEG-dioleoylmonoamine complexes is determined in vitro as follows. The siRNA used isa double stranded sequence of nucleotides intended to produce aknockdown of endogenous Caveolin-1 (Cav-1) transcript levels. Thecationic lipids used are mixtures of dioleoyl monoamine (Example 3) andmPEG-dioleoyl monoamine (Example 34). To prepare the liposomes, thelipids are dissolved and mixed in chloroform to assure a homogenousmixture. The organic solvent is removed by rotary evaporation yielding athin lipid film on the walls of a round-bottom flask. Chloroform isfurther evaporated by placing the round-bottom flask on a vacuum pumpovernight. The resulting lipid film is rehydrated with distilled waterat a desired concentration and vortexed vigorously for several minutes.The solution is placed in a bath sonicator for 1 hour and is thensterile syringe filtered through a 200 nm syringe filter to give thefinal liposomal solution. siRNA dissolved in water is added to theliposome solution. The resulting nanoparticles are suspended in 5%dextrose by adding an equal amount of 10% dextrose to the complexedsiRNA particles. SCCVII cells (0.5×10′ cells/well) are seeded into24-well tissue culture plates in 10% FBS. Each well is incubated for 6hours with 0.5 μg of complexed siRNA in absence or presence of FBS in atotal volume of 250 μL DMEM. When the incubation period is concluded forthe cells lacking FBS in their medium, 250 μL of DMEM supplemented with20% FBS is added to the transfected cells and incubated further foranother 40 hours. To cells with FBS in their transfection medium, 250 μLof DMEM supplemented with 10% FBS is added to the transfected cells andincubated further for another 40 hours. At the end of the incubationperiod, knockdown of Cav-transcript is measured in cell lysates. Toperform Cav-1 transcript analysis, cells are lysed using Tri-reagent andthen quantified for the level of Cav-1 transcript using a qRT-PCRdetection kit. Cav-1 transcript is inhibited up to 90% compared to anon-silencing control (FIG. 5A).

Example 42

Transfection activity of encapsulated siRNA with dioleoylmonoamine/mPEG-dioleoyl monoamine

The transfection activity of encapsulated siRNA with dioleoylmonoamine/mPEG-dioleoyl monoamine is determined in vitro as follows. ThesiRNA used is a double stranded sequence of nucleotides intended toproduce a knockdown of endogenous Caveolin-1 (Cav-1) transcript levels.The cationic lipids used are mixtures of dioleoyl monoamine (Example 3)and mPEG-dioleoyl monoamine (Example 34). To prepare the encapsulationliposomes, the lipids are dissolved and mixed in chloroform to assure ahomogenous mixture. The organic solvent is removed by rotary evaporationyielding a thin lipid film on the walls of a round-bottom flask.Chloroform is further evaporated by placing the round-bottom flask on avacuum pump overnight. The resulting lipid film is dissolved initiallyin 100% ethanol then brought to 50%. siRNA dissolved in water is addedto the liposomes in ethanol solution. Ethanol is evaporated from theliposomes/siRNA mixture by a rotary evaporation system. The resultingnanoparticles are suspended in 5% dextrose by adding an equal amount of10% dextrose to the encapsulated siRNA particles. SCCVII cells (0.5×10⁵cells/well) are seeded into 24-well tissue culture plates in 10% FBS.Each well is incubated for hours with 0.5 μg of encapsulated siRNA inabsence or presence of FBS in a total volume of 250 μL DMEM. When theincubation period is concluded for the cells lacking FBS in theirmedium, 250 μl of DMEM supplemented with 20% FBS is added to thetransfected cells and incubated further for another 40 hours. To cellswith FBS in their transfection medium, 250 μL of DMEM supplemented with10% FBS is added to the transfected cells and incubated further foranother 40 hours. At the end of the incubation period, knockdown ofCav-transcript is measured in the cell lysates. For Cav-1 transcriptanalysis, cells are lysed using Tri-reagent and then quantified for thelevel of VEGF transcript using a qRT-PCR detection kit. Cav-1 transcriptis inhibited up to 45% compared to a non-silencing control (See FIG.5B).

Example 43

Caveolin-1 transcript knockdown in lung following administration ofsiRNA complexed with dioleoyl monoamine and mPEG-dioleoyl monoamine

Female ICR mice (17-22 g) are injected intravenously (IV) with 200 μL ofpreviously formulated siRNA targeting Caveolin-1 (Cav-1) transcript.siRNA complexes contain 10:1 mixture of dioleoylmonoamine andmPEG-dioleoyl monoamine with 100, 60, 40, 20, or 10 μg of siRNA (20:1N:P ratio) (ref. Example 9). At 48 hours after injection the animals areeuthanized and lungs are harvested for target Cav-1 transcript analysisand siRNA quantification using qRT-PCR. The CAV-1 transcript levels(normalized to β-actin as an internal control) are expressed as apercent expression relative to untreated control animals. Resultsindicate a dose-dependent reduction of Cav-1 transcript levels in lungsof animals ranging from >60% in animals given 100 μg siRNA to ˜13% inanimals given 10 μg siRNA. Quantification of the injected siRNAindicates a dose dependent increase in the absolute amount of siRNAdistributed to the lungs (FIG. 6). At the lowest dose ˜5% of theinjected siRNA is distributed to the lungs. At the highest dose ˜50% ofthe injected siRNA is distributed to the lungs. The number of animals(“n”) is 6 for each group.

Example 44

Transfection activity of encapsulated siRNA with dioleoylmonoamine/lactobionyl-dioleoyl monoamine

The transfection activity of encapsulated siRNA with dioleoylmonoamine/lactobionyl-dioleoyl monoamine is determined in vitro asfollows. The siRNA used is a double stranded sequence of nucleotidesintended to produce a knockdown of β-actin transcript levels. Thecationic lipids used are mixtures of dioleoyl monoamine (Example 3) andlactobionyl-dioleoyl monoamine (Example 35). To prepare theencapsulation liposomes, the lipids are dissolved and mixed inchloroform to assure a homogenous mixture. The organic solvent isremoved by rotary evaporation yielding a thin lipid film on the walls ofa round-bottom flask. Chloroform is further evaporated by placing theround-bottom flask on a vacuum pump overnight. The resulting lipid filmis dissolved initially in 100% ethanol then brought to 50%. siRNAdissolved in water is added to the liposomes in ethanol solution.Ethanol is evaporated from the liposomes/siRNA mixture by a rotaryevaporation system. The resulting nanoparticles are suspended in 5%dextrose by adding an equal amount of 10% dextrose to the encapsulatedsiRNA particles. Hepa16 cells (0.5×10⁵ cells/well) are seeded into24-well tissue culture plates in 10% FBS. Each well is incubated forhours with 0.5 μg of encapsulated siRNA in absence or presence of FBS ina total volume of 250 μL DMEM. When the incubation period is concludedfor the cells lacking FBS in their medium, 250 μl of DMEM supplementedwith 20% FBS is added to the transfected cells and incubated further foranother 40 hours. To cells with FBS in their transfection medium, 250 μLof DMEM supplemented with 10% FBS is added to the transfected cells andincubated further for another 40 hours. At the end of the incubationperiod, knockdown of β-actin transcript is measured in the cell lysates.For transcript analysis, cells are lysed using Tri-reagent and thenquantified for the level of β-actin transcript using a qRT-PCR detectionkit. β-actin transcript is inhibited to 90% compared to a non-silencingcontrol, while samples containing identical particles lacking the LBAligand showed only 57% inhibition compared to a non-silencing control(FIG. 7).

Example 45

Controlled release of siRNA complexed with dioleoyl monoamine fromcrosslinked gel

siRNA complexed with dioleoyl monoamine is formulated as described inExample 39 containing 50 μg Cav-1 siRNA and 1.4 μg dioleoyl monoamine ina volume of 50 μL. Sodium alginate (Type A) is dissolved in water togive a 2% solution. An alginate solution (100 μL) is added to a singlewell of a 96-well plate followed by 50 μL of the dioleoylmonoamine/Cav-siRNA solution. After thorough mixing, 50 μL of a 0.68 MCaCl₂ solution is added to the previous mixture to crosslink the gel.One hundred μL of water or 0.1 M EDTA (which serves to break down thegel by complexing the Ca²⁺ ions) is then added to ensure completesubmersion of the gel in aqueous solution. At the prescribed timepoints, the supernatants are removed (200 μL) and replaced with fresh200 μL water or 0.1 M EDTA. After 84 hours, all collected samples areassayed for Cav-1 siRNA content using qRT-PCR. The siRNA amountscollected at each time point are summed in order to determine amountreleased over time. In samples that have the addition of EDTA, 100% ofthe loaded dioleoyl monoamine formulated siRNA is released after 84hours. In samples which do not contain EDTA, a significantly slowerrelease kinetic was observed with <20% of the total siRNA loaded beingreleased by 84 hours (FIG. 8).

Example 46

Caveolin-1 transcript knockdown in lung and liver followingadministration of siRNA complexed with dioleoyl monoamine andmPEG-dioleoyl monoamine in comparison to siRNA complexed with othercommercially available cationic lipid and cationic polymeric systems

Female ICR mice (17-22 g) are injected intravenously (IV) with 200 μL ofa previously formulated siRNA targeting Caveolin-1 (Cav-1) transcript.siRNA complexes contain 10:1 mixture of dioleoyl monoamine andmPEG-dioleoyl monoamine with 40 μg siRNA (20:1 N:P ratio) (ref. Example39). In addition, siRNA is formulated with either DOTAP:DOPE (1:1) at a20:1 N:P ratio, or with 25 kDa branched PEI (10:1). A total of 40 μgsiRNA formulated with DOTAP:DOPE (in 200 μL) is injected IV into mice ora total of 20 μg of siRNA formulated with branched PEI (in 100 μL) isinjected IV. Branched PEI is used in a lower amount in order to try andmitigate the known toxicities associated with this formulation. At 48hours after injection animals are euthanized and lungs and livers areharvested for target Cav-1 transcript analysis and siRNA quantificationusing qRT-PCR. The CAV-1 transcript levels (normalized to [3-actin as aninternal control) are expressed as a percent expression relative tountreated control animals. The number of animals (“n”) is 5 for eachgroup. Results indicate significant (˜60%) transcript knockdown in CAV-1expression in lung tissue and a ˜33% transcript knockdown in liver (FIG.9). No significant transcript knockdown was noted for the DOTAP:DOPEformulated siRNA. For the branched PEI formulated siRNA the animals dieddue to toxicity associated with the formulation prior to tissue harvestand could therefore not be analyzed; the dioleoylmonoamine/mPEG-dioleoyl monoamine formulated siRNA and the DOTAP:DOPEformulated siRNA were administered with little toxicity.

Example 47

Transfection activity of encapsulated siRNA with dioleoylcrossamine/PSMA targeting aptamer

The transfection activity of encapsulated dsRNA with dioleoylcrossamine/PSMA targeting aptamer is determined in vitro as follows. ThesiRNA used is a double stranded sequence of nucleotides intended toproduce a knockdown of Cav-1 transcript levels. The cationic lipid usedis dioleoyl crossamine (Example 1). The RNA aptamer targets the prostatespecific membrane antigen and includes a ssRNA uridine tail on the 3′end (PSMA aptamer sequence: 5′GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUAAUUUUUUUUUUUUUUUUUUUU 3′). Anon-binding mutant aptamer is included as a negative control (mutantPSMA aptamer sequence: 5′GGGAGGACGAUGCGGAUCAGCCAUCCUUACGUCACUCCUAAUUUUUUUUUUUUUUUUUUUU 3′).Encapsulated liposomes are prepared as described in Example 45. Targetedliposomes are prepared by adding the RNA aptamer to the encapsulatedliposomes and incubating the mixture at room temperature for 30 minutes.LNCaP cells (2.5×10⁵ cells/well) are seeded into 12 well tissue cultureplates in 10% FBS. Each well is incubated for 5 hours with 0.1 μg oftargeted liposomes or non-targeted control liposomes (complexed with anon-binding mutant aptamer) in the absence of FBS in a total volume of500 μL RPMI 1640. When the incubation period is concluded, the media isreplaced with 500 of RPMI 1640 supplemented with 10% FBS and incubatedfor an additional 40 hours. At the end of the incubation period,knockdown of the Cav-1 transcript is measured in the cell lysates. Fortranscript analysis, cells are lysed and total RNA is purified usingQiagen's RNEasy kit (Qiagen product number: 74106). Transcript levelsfor Cav-1 and GAPDH (internal control) were quantified using a qRT-PCRdetection kit. Cav-1 transcript levels are depleted in cells treatedwith PSMA targeting liposomes by 70% compared to non-silencing control.This is in contrast to cells treated with the non-targeting mutant PSMAaptamer, which show no depletion of Cav-transcripts compared tonon-silencing controls. Treatment of cells lacking the prostate specificmembrane antigen (Chinese hamster ovary cells) show similar levels ofCav-1 depletion (˜35%) compared to non-silencing controls independent oftargeting aptamer identity (FIG. 10)

The invention and the manner and process of making and using it, are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the invention and that modifications may be made thereinwithout departing from the spirit or scope of the invention as set forthin the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thisspecification.

1-47. (canceled)
 48. A compound of the formula:

n1, n2, and n3 are independently 1, 2, 3, or 4; X and X′ areindependently a bond, oxygen, or nitrogen; R₁ and R₂ are independentlyC₈-C₂₅ hydrocarbon groups optionally containing from 1-4 double ortriple bonds; and G is hydrogen or a polymer moiety.
 49. A compoundaccording to claim 48, wherein n1, n2, n3, and n4 are all 1, and X andX′ are bonds. 50-54. (canceled)
 55. A compound according to claim 48,wherein R₁ and R₂ are independently C₁₄-C₂₀ hydrocarbon groupscontaining 1 double bond.
 56. A compound according to claim 55, whereinboth of —C(O)X—R₁ and —C(O)X′—R₂ represent oleoyl groups. 57-58.(canceled)
 59. A compound according to claim 48, wherein the G is apolyoxyalkylene, polyvinylpyrrolidone, polyacrylamide,polydimethylacrylamide, polyvinyl alcohol, dextran, poly (L-glutamicacid), styrene maleic anhydride, poly-N-(2-hydroxypropyl)methacrylamide, or polydivinylether maleic anhydride.
 60. A compoundaccording to claim 59, wherein the polymer comprises at least one linkergroup between polymer units.
 61. A compound according to claim 59, wherethe polymer moiety is a polyoxyalkylene. 62-66. (canceled)
 67. Acompound of the formula:

wherein n1, n2, and n3 are independently 1, 2, 3, or 4; X and X′ areindependently a bond, oxygen, or nitrogen; R₁ and R₂ are independentlyC₈-C₂₅ hydrocarbon groups optionally containing from 1-4 double ortriple bonds; and T is a targeting ligand; and G is a bond, or a polymermoiety. 68-83. (canceled)
 84. A formulation comprising a compound ofclaim 48 and a molecule selected from the group consisting of: (a)ribosomal RNA; antisense polynucleotides of RNA or DNA; aptamers;ribozymes; siRNA; shRNA; miRNA; and polynucleotides of genomic DNA,cDNA, or mRNA that encode for a therapeutically useful protein; or (c)proteins, peptides, cholesterol, hormones, small molecule pharmaceuticalcompounds, vitamins, and co-factors.
 85. A formulation according toclaim 84, where the formulation comprises particles formed by thecompound and the molecule.
 86. A formulation according to claim 85,where the particles have a median diameter of less than about 500 nm.87. (canceled)
 88. A method of modulating expression of a targetsequence, said method comprising administering to a mammalian subject atherapeutically effective amount of a formulation of claim
 84. 89-93.(canceled)
 94. A method for modulating expression of a gene in a cell,comprising contacting the cell with a formulation of claim
 84. 95-104.(canceled)
 105. A formulation comprising a compound of formula A and acompound of Formula B

wherein n1, n2, and n3 are independently 1, 2, 3, or 4; X and X′ areindependently a bond, oxygen, or nitrogen; R₁ and R₂ are independentlyC₈-C₂₅ hydrocarbon groups optionally containing from 1-4 double ortriple bonds; and in Formula A, G is hydrogen; and in Formula B, G is apolymer moiety; and a molecule selected from the group consisting of:(a) ribosomal RNA; antisense polynucleotides of RNA or DNA; aptamers;ribozymes; siRNA; shRNA; miRNA; and polynucleotides of genomic DNA,cDNA, or mRNA that encode for a therapeutically useful protein.
 106. Amethod for in vivo delivery of siRNA or plasmid DNA to a mammaliansubject comprising administering to the subject a formulation of claim105.
 107. A method for treating a disease in a mammalian subjectcomprising administering to the subject a formulation of claim
 105. 108.A method for treating a disease characterized by unregulated cell growthin a subject comprising administering to the subject a formulation ofclaim
 105. 109. A method for treating a disease in lung tissue in asubject comprising administering to the subject a formulation of claim105.
 110. A method according to claim 108, where the disease is cancer.111. A formulation according to claim 105, wherein the ratio of thecompound of Formula A to the compound of Formula B in the formulation is1:1 to 10:1.
 112. A composition comprising a compound of formula A and acompound of Formula B

wherein n1, n2, and n3 are independently 1, 2, 3, or 4; X and X′ areindependently a bond, oxygen, or nitrogen; R₁ and R₂ are independentlyC₈-C₂₅ hydrocarbon groups optionally containing from 1-4 double ortriple bonds; and in Formula A, G is hydrogen; and in Formula B, G is apolymer moiety.
 113. A formulation according to claim 112, wherein theratio of the compound of Formula A to the compound of Formula B in theformulation is 1:1 to 10:1.